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Rossella MANFREDINI
Professore Ordinario
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze - sede Centro di Medicina Rigenerativa S.Ferrari
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Pubblicazioni
2023
- Antihypertensive treatment changes and related clinical outcomes in older hospitalized patients
[Articolo su rivista]
Cicco, S.; D'Abbondanza, M.; Proietti, M.; Zaccone, V.; Pes, C.; Caradio, F.; Mattioli, M.; Piano, S.; Marra, A. M.; Nobili, A.; Mannucci, P. M.; Pietrangelo, A.; Sesti, G.; Buzzetti, E.; Salzano, A.; Cimellaro, A.; Mannucci, P. M.; Nobili, A.; Sesti, G.; Pietrangelo, A.; Perticone, F.; Violi, F.; Corazza, G. R.; Corrao, S.; Marengoni, A.; Salerno, F.; Cesari, M.; Tettamanti, M.; Pasina, L.; Franchi, C.; Franchi, C.; Novella, A.; Tettamanti, M.; Miglio, G.; Tettamanti, M.; Galbussera, A. A.; Ardoino, I.; Novella, A.; Prisco, D.; Silvestri, E.; Emmi, G.; Bettiol, A.; Mattioli, I.; Biolo, G.; Zanetti, M.; Bartelloni, G.; Zaccari, M.; Chiuch, M.; Vanoli, M.; Grignani, G.; Pulixi, E. A.; Pirro, M.; Lupattelli, G.; Bianconi, V.; Alcidi, R.; Giotta, A.; Mannarino, M. R.; Girelli, D.; Busti, F.; Marchi, G.; Barbagallo, M.; Dominguez, L.; Beneduce, V.; Cacioppo, F.; Corrao, S.; Natoli, G.; Mularo, S.; Raspanti, M.; Argano, C.; Cavallaro, F.; Zoli, M.; Matacena, M. L.; Orio, G.; Magnolfi, E.; Serafini, G.; Simili, A.; Brunori, M.; Lazzari, I.; Simili, A.; Cappellini, M. D.; Fabio, G.; De Amicis, M. M.; De Luca, G.; Scaramellini, N.; Di Stefano, V.; Leoni, S.; Seghezzi, S.; Di Mauro, A. D.; Maira, D.; Mancarella, M.; Lucchi, T.; Rossi, P. D.; Clerici, M.; Leoni, S.; Di Mauro, A. D.; Bonini, G.; Conti, F.; Prolo, S.; Fabrizi, M.; Martelengo, M.; Vigani, G.; Di Sabatino, A.; Miceli, E.; Lenti, M. V.; Pisati, M.; Pitotti, L.; Padula, D.; Antoci, V.; Cambie, G.; Pontremoli, R.; Beccati, V.; Nobili, G.; Leoncini, G.; Alberto, J.; Cattaneo, F.; Anastasio, L.; Sofia, L.; Carbone, M.; Cipollone, F.; Guagnano, M. T.; Rossi, I.; Valeriani, E.; D'Ardes, D.; Esposito, L.; Sestili, S.; Angelucci, E.; Mancuso, G.; Calipari, D.; Bartone, M.; Delitala, G.; Berria, M.; Delitala, A.; Muscaritoli, M.; Molfino, A.; Petrillo, E.; Giorgi, A.; Gracin, C.; Imbimbo, G.; Zuccala, G.; D'Aurizio, G.; Romanelli, G.; Marengoni, A.; Volpini, A.; Lucente, D.; Manzoni, F.; Pirozzi, A.; Zucchelli, A.; Picardi, A.; Gentilucci, U. V.; Gallo, P.; Dell'Unto, C.; Bellelli, G.; Corsi, M.; Antonucci, C.; Sidoli, C.; Principato, G.; Bonfanti, A.; Szabo, H.; Mazzola, P.; Piazzoli, A.; Corsi, M.; Arturi, F.; Succurro, E.; Tassone, B.; Giofre, F.; Serra, M. G.; Bleve, M. A.; Brucato, A.; De Falco, T.; Negro, E.; Brenna, M.; Trotta, L.; Squintani, G. L.; Randi, M. L.; Fabris, F.; Bertozzi, I.; Bogoni, G.; Rabuini, M. V.; Prandini, T.; Ratti, F.; Zurlo, C.; Cerruti, L.; Cosi, E.; Manfredini, R.; Fabbian, F.; Boari, B.; De Giorgi, A.; Tiseo, R.; Paolisso, G.; Rizzo, M. R.; Catalano, C.; Di Meo, I.; Borghi, C.; Strocchi, E.; Ianniello, E.; Soldati, M.; Schiavone, S.; Bragagni, A.; Leoni, F. G.; De Sando, V.; Scarduelli, S.; Cammarosano, M.; Pareo, I.; Sabba, C.; Vella, F. S.; Suppressa, P.; De Vincenzo, G. M.; Comitangelo, A.; Amoruso, E.; Custodero, C.; Re, G.; Schilardi, A.; Loparco, F.; Fenoglio, L.; Falcetta, A.; Giraudo, A. V.; D'Aniano, S.; Fracanzani, A. L.; Tiraboschi, S.; Cespiati, A.; Oberti, G.; Sigon, G.; Cinque, F.; Peyvandi, F.; Rossio, R.; Colombo, G.; Agosti, P.; Pagliaro, E.; Semproni, E.; Ciro, C.; Monzani, V.; Savojardo, V.; Ceriani, G.; Folli, C.; Salerno, F.; Pallini, G.; Montecucco, F.; Ottonello, L.; Caserza, L.; Vischi, G.; Kassem, S.; Liberale, L.; Liberato, N. L.; Tognin, T.; Purrello, F.; Di Pino, A.; Piro, S.; Rozzini, R.; Falanga, L.; Pisciotta, M. S.; Bellucci, F. B.; Buffelli, S.; Ferrandina, C.; Mazzeo, F.; Spazzini, E.; Cono, G.; Cesaroni, G.; Montrucchio, G.; Peasso, P.; Favale, E.; Poletto, C.; Margaria, C.; Sanino, M.; Violi, F.; Perri, L.; Guasti, L.; Rotunno, F.; Castiglioni, L.; Maresca, A.; Squizzato, A.; Campiotti, L.; Grossi, A.; Diprizio, R. D.; Dentali, F.; Bertolotti, M.; Mussi, C.; Lancellotti, G.; Libbra, M. V.; Galassi, M.; Grassi, Y.; Greco, A.; Bigi, E.; Pellegrini, E.; Orlandi, L.; Dondi, G.; Carulli, L.; Sciacqua, A.; Perticone, M.; Battaglia, R.; Maio, R.; Scozzafava, A.; Condoleo, V.; Falbo, T.; Colangelo, L.; Filice, M.; Clausi, E
abstract
Background: Hypertension management in older patients represents a challenge, particularly when hospitalized. Objective: The objective of this study is to investigate the determinants and related outcomes of antihypertensive drug prescription in a cohort of older hospitalized patients. Methods: A total of 5671 patients from REPOSI (a prospective multicentre observational register of older Italian in-patients from internal medicine or geriatric wards) were considered; 4377 (77.2%) were hypertensive. Minimum treatment (MT) for hypertension was defined according to the 2018 ESC guidelines [an angiotensin-converting-enzyme-inhibitor (ACE-I) or an angiotensin-receptor-blocker (ARB) with a calcium-channel-blocker (CCB) and/or a thiazide diuretic; if >80 years old, an ACE-I or ARB or CCB or thiazide diuretic]. Determinants of MT discontinuation at discharge were assessed. Study outcomes were any cause rehospitalization/all cause death, all-cause death, cardiovascular (CV) hospitalization/death, CV death, non-CV death, evaluated according to the presence of MT at discharge. Results: Hypertensive patients were older than normotensives, with a more impaired functional status, higher burden of comorbidity and polypharmacy. A total of 2233 patients were on MT at admission, 1766 were on MT at discharge. Discontinuation of MT was associated with the presence of comorbidities (lower odds for diabetes, higher odds for chronic kidney disease and dementia). An adjusted multivariable logistic regression analysis showed that MT for hypertension at discharge was associated with lower risk of all-cause death, all-cause death/hospitalization, CV death, CV death/hospitalization and non-CV death. Conclusions: Guidelines-suggested MT for hypertension at discharge is associated with a lower risk of adverse clinical outcomes. Nevertheless, changes in antihypertensive treatment still occur in a significant proportion of older hospitalized patients.
2023
- Characteristics and clinical behavior of acute myeloid leukemia harboring rare non-A/B/D nucleophosmin (NPM1) gene mutation subtypes: a single-center experience and review of the literature
[Articolo su rivista]
Mutti, M.; Cordella, S.; Parisotto, A.; Bettelli, F.; Morselli, M.; Cuoghi, A.; Bresciani, P.; Messerotti, A.; Gilioli, A.; Pioli, V.; Giusti, D.; Colaci, E.; Cassanelli, L.; Paolini, A.; Martinelli, S.; Maffei, R.; Riva, G.; Nasillo, V.; Sarti, M.; Trenti, T.; Comoli, P.; Tagliafico, E.; Manfredini, R.; Eccher, A.; Lagreca, I.; Barozzi, P.; Potenza, L.; Marasca, R.; Candoni, A.; Luppi, M.; Forghieri, F.
abstract
2023
- Chromosome 9p Duplication Promotes T-Cell Exhaustion and Enhances Stem Cell Clonogenic Potential in JAK2-Mutant Myeloproliferative Neoplasms
[Abstract in Rivista]
Norfo, Ruggiero; Carretta, Chiara; Parenti, Sandra; Badii, Filippo; Bertesi, Matteo; Rontauroli, Sebastiano; Tavernari, Lara; Genovese, Elena; Sperduti, Samantha; Enzo, Elena; Mirabile, Margherita; Pedrazzi, Francesca; Pessina, Chiara; Colugnat, Ilaria; Mora, Barbara; Maccaferri, Monica; Tenedini, Elena; Martinelli, Silvia; Bianchi, Elisa; Casarini, Livio; Potenza, Leonardo; Luppi, Mario; Tagliafico, Enrico; Guglielmelli, Paola; Simoni, Manuela; Passamonti, Francesco; Vannucchi, Alessandro Maria; Manfredini, Rossella
abstract
2023
- Comparison between Cultivated Oral Mucosa and Ocular Surface Epithelia for COMET Patients Follow-Up
[Articolo su rivista]
Attico, Eustachio; Galaverni, Giulia; Torello, Andrea; Bianchi, Elisa; Bonacorsi, Susanna; Losi, Lorena; Manfredini, Rossella; Lambiase, Alessandro; Rama, Paolo; Pellegrini, Graziella
abstract
Total bilateral Limbal Stem Cell Deficiency is a pathologic condition of the ocular surface due to the loss of corneal stem cells. Cultivated oral mucosa epithelial transplantation (COMET) is the only autologous successful treatment for this pathology in clinical application, although abnormal peripheric corneal vascularization often occurs. Properly characterizing the regenerated ocular surface is needed for a reliable follow-up. So far, the univocal identification of transplanted oral mucosa has been challenging. Previously proposed markers were shown to be co-expressed by different ocular surface epithelia in a homeostatic or perturbated environment. In this study, we compared the transcriptome profile of human oral mucosa, limbal and conjunctival cultured holoclones, identifying Paired Like Homeodomain 2 (PITX2) as a new marker that univocally distinguishes the transplanted oral tissue from the other epithelia. We validated PITX2 at RNA and protein levels to investigate 10-year follow-up corneal samples derived from a COMET-treated aniridic patient. Moreover, we found novel angiogenesis-related factors that were differentially expressed in the three epithelia and instrumental in explaining the neovascularization in COMET-treated patients. These results will support the follow-up analysis of patients transplanted with oral mucosa and provide new tools to understand the regeneration mechanism of transplanted corneas.
2023
- Inhibition of ERK1/2 signaling prevents bone marrow fibrosis by reducing osteopontin plasma levels in a myelofibrosis mouse model
[Articolo su rivista]
Bianchi, Elisa; Rontauroli, Sebastiano; Tavernari, Lara; Mirabile, Margherita; Pedrazzi, Francesca; Genovese, Elena; Sartini, Stefano; Dall'Ora, Massimiliano; Grisendi, Giulia; Fabbiani, Luca; Maccaferri, Monica; Carretta, Chiara; Parenti, Sandra; Fantini, Sebastian; Bartalucci, Niccolò; Calabresi, Laura; Balliu, Manjola; Guglielmelli, Paola; Potenza, Leonardo; Tagliafico, Enrico; Losi, Lorena; Dominici, Massimo; Luppi, Mario; Vannucchi, Alessandro Maria; Manfredini, Rossella
abstract
Clonal myeloproliferation and development of bone marrow (BM) fibrosis are the major pathogenetic events in myelofibrosis (MF). The identification of novel antifibrotic strategies is of utmost importance since the effectiveness of current therapies in reverting BM fibrosis is debated. We previously demonstrated that osteopontin (OPN) has a profibrotic role in MF by promoting mesenchymal stromal cells proliferation and collagen production. Moreover, increased plasma OPN correlated with higher BM fibrosis grade and inferior overall survival in MF patients. To understand whether OPN is a druggable target in MF, we assessed putative inhibitors of OPN expression in vitro and identified ERK1/2 as a major regulator of OPN production. Increased OPN plasma levels were associated with BM fibrosis development in the Romiplostim-induced MF mouse model. Moreover, ERK1/2 inhibition led to a remarkable reduction of OPN production and BM fibrosis in Romiplostim-treated mice. Strikingly, the antifibrotic effect of ERK1/2 inhibition can be mainly ascribed to the reduced OPN production since it could be recapitulated through the administration of anti-OPN neutralizing antibody. Our results demonstrate that OPN is a novel druggable target in MF and pave the way to antifibrotic therapies based on the inhibition of ERK1/2-driven OPN production or the neutralization of OPN activity.
2023
- Prognostic Relevance of Multi-Antigenic Myeloma-Specific T-Cell Assay in Patients with Monoclonal Gammopathies
[Articolo su rivista]
Lagreca, Ivana; Nasillo, Vincenzo; Barozzi, Patrizia; Castelli, Ilaria; Basso, Sabrina; Castellano, Sara; Paolini, Ambra; Maccaferri, Monica; Colaci, Elisabetta; Vallerini, Daniela; Natali, Patrizia; Debbia, Daria; Pirotti, Tommaso; Ottomano, Anna Maria; Maffei, Rossana; Bettelli, Francesca; Giusti, Davide; Messerotti, Andrea; Gilioli, Andrea; Pioli, Valeria; Leonardi, Giovanna; Forghieri, Fabio; Bresciani, Paola; Cuoghi, Angela; Morselli, Monica; Manfredini, Rossella; Longo, Giuseppe; Candoni, Anna; Marasca, Roberto; Potenza, Leonardo; Tagliafico, Enrico; Trenti, Tommaso; Comoli, Patrizia; Luppi, Mario; Riva, Giovanni
abstract
: Multiple Myeloma (MM) typically originates from underlying precursor conditions, known as Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). Validated risk factors, related to the main features of the clonal plasma cells, are employed in the current prognostic models to assess long-term probabilities of progression to MM. In addition, new prognostic immunologic parameters, measuring protective MM-specific T-cell responses, could help to identify patients with shorter time-to-progression. In this report, we described a novel Multi-antigenic Myeloma-specific (MaMs) T-cell assay, based on ELISpot technology, providing simultaneous evaluation of T-cell responses towards ten different MM-associated antigens. When performed during long-term follow-up (mean 28 months) of 33 patients with either MGUS or SMM, such deca-antigenic myeloma-specific immunoassay allowed to significantly distinguish between stable vs. progressive disease (p < 0.001), independently from the Mayo Clinic risk category. Here, we report the first clinical experience showing that a wide (multi-antigen), standardized (irrespective to patients' HLA), MM-specific T-cell assay may routinely be applied, as a promising prognostic tool, during the follow-up of MGUS/SMM patients. Larger studies are needed to improve the antigenic panel and further explore the prognostic value of MaMs test in the risk assessment of patients with monoclonal gammopathies.
2022
- BTK Inhibitors Impair Platelet-Mediated Antifungal Activity
[Articolo su rivista]
Nasillo, V.; Lagreca, I.; Vallerini, D.; Barozzi, P.; Riva, G.; Maccaferri, M.; Paolini, A.; Forghieri, F.; Fiorcari, S.; Maffei, R.; Martinelli, S.; Atene, C. G.; Castelli, I.; Marasca, R.; Potenza, L.; Comoli, P.; Manfredini, R.; Tagliafico, E.; Trenti, T.; Luppi, M.
abstract
In recent years, the introduction of new drugs targeting Bruton’s tyrosine kinase (BTK) has allowed dramatic improvement in the prognosis of patients with chronic lymphocytic leukemia (CLL) and other B-cell neoplasms. Although these small molecules were initially considered less immunosuppressive than chemoimmunotherapy, an increasing number of reports have described the occurrence of unexpected opportunistic fungal infections, in particular invasive aspergillosis (IA). BTK represents a crucial molecule in several signaling pathways depending on different immune receptors. Based on a variety of specific off-target effects on innate immunity, namely on neutrophils, monocytes, pulmonary macrophages, and nurse-like cells, ibrutinib has been proposed as a new host factor for the definition of probable invasive pulmonary mold disease. The role of platelets in the control of fungal growth, through granule-dependent mechanisms, was described in vitro almost two decades ago and is, so far, neglected by experts in the field of clinical management of IA. In the present study, we confirm the antifungal role of platelets, and we show, for the first time, that the exposure to BTK inhibitors impairs several immune functions of platelets in response to Aspergillus fumigatus, i.e., the ability to adhere to conidia, activation (as indicated by reduced expression of P-selectin), and direct killing activity. In conclusion, our experimental data suggest that antiplatelet effects of BTK inhibitors may contribute to an increased risk for IA in CLL patients.
2022
- Novel Molecular Insights into Leukemic Evolution of Myeloproliferative Neoplasms: A Single Cell Perspective
[Articolo su rivista]
Rontauroli, Sebastiano; Carretta, Chiara; Parenti, Sandra; Bertesi, Matteo; Manfredini, Rossella
abstract
Myeloproliferative neoplasms (MPNs) are clonal disorders originated by the serial acquisition of somatic mutations in hematopoietic stem/progenitor cells. The major clinical entities are represented by polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), that are caused by driver mutations affecting JAK2, MPL or CALR. Disease progression is related to molecular and clonal evolution. PV and ET can progress to secondary myelofibrosis (sMF) but can also evolve to secondary acute myeloid leukemia (sAML). PMF is associated with the highest frequency of leukemic transformation, which represents the main cause of death. sAML is associated with a dismal prognosis and clinical features that differ from those of de novo AML. The molecular landscape distinguishes sAML from de novo AML, since the most frequent hits involve TP53, epigenetic regulators, spliceosome modulators or signal transduction genes. Single cell genomic studies provide novel and accurate information about clonal architecture and mutation acquisition order, allowing the reconstruction of clonal dynamics and molecular events that accompany leukemic transformation. In this review, we examine our current understanding of the genomic heterogeneity in MPNs and how it affects disease progression and leukemic transformation. We focus on molecular events elicited by somatic mutations acquisition and discuss the emerging findings coming from single cell studies.
2022
- SOX2 Is a Univocal Marker for Human Oral Mucosa Epithelium Useful in Post-COMET Patient Characterization
[Articolo su rivista]
Attico, Eustachio; Galaverni, Giulia; Bianchi, Elisa; Losi, Lorena; Manfredini, Rossella; Lambiase, Alessandro; Rama, Paolo; Pellegrini, Graziella
abstract
Total bilateral Limbal Stem Cells Deficiency is a pathologic condition of the ocular surface due to loss or impairment of corneal stem cell function, altering homeostasis of the corneal epithelium. Cultivated Oral Mucosa Epithelial Transplantation (COMET) is the only autologous treatment for this pathology. During the follow-up, a proper characterization of the transplanted oral mucosa on the ocular surface supports understanding the regenerative process. The previously proposed markers for oral mucosa identification (e.g., keratins 3 and 13) are co-expressed by corneal and conjunctival epithelia. Here, we propose a new specific marker to distinguish human oral mucosa from the epithelia of the ocular surface. We compared the transcriptome of holoclones (stem cells) from the human oral mucosa, limbal and conjunctival cultures by microarray assay. High expression of SOX2 identified the oral mucosa vs. cornea and conjunctiva, while PAX6 was highly expressed in corneal and conjunctival epithelia. The transcripts were validated by qPCR, and immunological methods identified the related proteins. Finally, the proposed markers were used to analyze a 10-year follow-up aniridic patient treated by COMET. These findings will support the follow-up analysis of COMET treated patients and help to shed light on the mechanism of corneal repair and regeneration.
2022
- The Response to Oxidative Damage Correlates with Driver Mutations and Clinical Outcome in Patients with Myelofibrosis
[Articolo su rivista]
Genovese, E.; Mirabile, M.; Rontauroli, S.; Sartini, S.; Fantini, S.; Tavernari, L.; Maccaferri, M.; Guglielmelli, P.; Bianchi, E.; Parenti, S.; Carretta, C.; Mallia, S.; Castellano, S.; Colasante, C.; Balliu, M.; Bartalucci, N.; Palmieri, R.; Ottone, T.; Mora, B.; Potenza, L.; Passamonti, F.; Voso, M. T.; Luppi, M.; Vannucchi, A. M.; Tagliafico, E.; Manfredini, R.
abstract
Myelofibrosis (MF) is the Philadelphia-negative myeloproliferative neoplasm characterized by the worst prognosis and no response to conventional therapy. Driver mutations in JAK2 and CALR impact on JAK-STAT pathway activation but also on the production of reactive oxygen species (ROS). ROS play a pivotal role in inflammation-induced oxidative damage to cellular components including DNA, therefore leading to greater genomic instability and promoting cell transformation. In order to unveil the role of driver mutations in oxidative stress, we assessed ROS levels in CD34+ hematopoietic stem/progenitor cells of MF patients. Our results demonstrated that ROS production in CD34+ cells from CALR-mutated MF patients is far greater compared with patients harboring JAK2 mutation, and this leads to increased oxidative DNA damage. Moreover, CALR-mutant cells show less superoxide dismutase (SOD) antioxidant activity than JAK2-mutated ones. Here, we show that high plasma levels of total antioxidant capacity (TAC) correlate with detrimental clinical features, such as high levels of lactate dehydrogenase (LDH) and circulating CD34+ cells. Moreover, in JAK2-mutated patients, high plasma level of TAC is also associated with a poor overall survival (OS), and multivariate analysis demonstrated that high TAC classification is an independent prognostic factor allowing the identification of patients with inferior OS in both DIPSS lowest and highest categories. Altogether, our data suggest that a different capability to respond to oxidative stress can be one of the mechanisms underlying disease progression of myelofibrosis.
2022
- The Role of T Cell Immunity in Monoclonal Gammopathy and Multiple Myeloma: From Immunopathogenesis to Novel Therapeutic Approaches
[Articolo su rivista]
Lagreca, I.; Riva, G.; Nasillo, V.; Barozzi, P.; Castelli, I.; Basso, S.; Bettelli, F.; Giusti, D.; Cuoghi, A.; Bresciani, P.; Messerotti, A.; Gilioli, A.; Pioli, V.; Colasante, C.; Vallerini, D.; Paolini, A.; Maccaferri, M.; Donatelli, F.; Forghieri, F.; Morselli, M.; Colaci, E.; Leonardi, G.; Marasca, R.; Potenza, L.; Manfredini, R.; Tagliafico, E.; Trenti, T.; Comoli, P.; Luppi, M.
abstract
Multiple Myeloma (MM) is a malignant growth of clonal plasma cells, typically arising from asymptomatic precursor conditions, namely monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Profound immunological dysfunctions and cyto-kine deregulation are known to characterize the evolution of the disease, allowing immune escape and proliferation of neoplastic plasma cells. In the past decades, several studies have shown that the immune system can recognize MGUS and MM clonal cells, suggesting that anti-myeloma T cell immunity could be harnessed for therapeutic purposes. In line with this notion, chimeric antigen receptor T cell (CAR-T) therapy is emerging as a novel treatment in MM, especially in the re-lapsed/refractory disease setting. In this review, we focus on the pivotal contribution of T cell im-pairment in the immunopathogenesis of plasma cell dyscrasias and, in particular, in the disease progression from MGUS to SMM and MM, highlighting the potentials of T cell-based immunother-apeutic approaches in these settings.
2021
- Activated IL-6 signaling contributes to the pathogenesis of, and is a novel therapeutic target for, CALR-mutated MPNs
[Articolo su rivista]
Balliu, M.; Calabresi, L.; Bartalucci, N.; Romagnoli, S.; Maggi, L.; Manfredini, R.; Lulli, M.; Guglielmelli, P.; Vannucchi, A. M.
abstract
Calreticulin (CALR), an endoplasmic reticulum–associated chaperone, is frequently mutated in myeloproliferative neoplasms (MPNs). Mutated CALR promotes downstream JAK2/STAT5 signaling through interaction with, and activation of, the thrombopoietin receptor (MPL). Here, we provide evidence of a novel mechanism contributing to CALR-mutated MPNs, represented by abnormal activation of the interleukin 6 (IL-6)-signaling pathway. We found that UT7 and UT7/mpl cells, engineered by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to express the CALR type 1–like (DEL) mutation, acquired cytokine independence and were primed to the megakaryocyte (Mk) lineage. Levels of IL-6 messenger RNA (mRNA), extracellular-released IL-6, membrane-associated glycoprotein 130 (gp130), and IL-6 receptor (IL-6R), phosphorylated JAK1 and STAT3 (p-JAK1 and p-STAT3), and IL-6 promoter region occupancy by STAT3 all resulted in increased CALR DEL cells in the absence of MPL stimulation. Wild-type, but not mutated, CALR physically interacted with gp130 and IL-6R, downregulating their expression on the cell membrane. Agents targeting gp130 (SC-144), IL-6R (tocilizumab [TCZ]), and cell-released IL-6 reduced proliferation of CALR DEL as well as CALR knockout cells, supporting a mutated CALR loss-of-function model. CD341 cells from CALR-mutated patients showed increased levels of IL-6 mRNA and p-STAT3, and colony-forming unit–Mk growth was inhibited by either SC144 or TCZ, as well as an IL-6 antibody, supporting cell-autonomous activation of the IL-6 pathway. Targeting IL-6 signaling also reduced colony formation by CD341 cells of JAK2V617F-mutated patients. The combination of TCZ and ruxolitinib was synergistic at very low nanomolar concentrations. Overall, our results suggest that target inhibition of IL-6 signaling may have therapeutic potential in CALR, and possibly JAK2V617F, mutated MPNs.
2021
- Automated capture-based NGS workflow: one thousand patients experience in a clinical routine framework
[Articolo su rivista]
Tenedini, E; Celestini, F; Iapicca, P; Marino, M; Castellano, S; Artuso, L; Biagiarelli, F; Cortesi, L; Toss, A; Barbieri, E; Roncucci, L; Pedroni, M; Manfredini, R; Luppi, M; Trenti, T; Tagliafico, E
abstract
Objectives: The Next Generation Sequencing (NGS) based mutational study of hereditary cancer genes is crucial to design tailored prevention strategies in subjects with different hereditary cancer risk. The ease of amplicon-based NGS library construction protocols contrasts with the greater uniformity of enrichment provided by capture-based protocols and so with greater chances for detecting larger genomic rearrangements and copy-number variations. Capture-based protocols, however, are characterized by a higher level of complexity of sample handling, extremely susceptible to human bias. Robotics platforms may definitely help dealing with these limits, reducing hands-on time, limiting random errors and guaranteeing process standardization.Methods: We implemented the automation of the CE-IVD SOPHiA Hereditary Cancer Solution (TM) (HCS) libraries preparation workflow by SOPHiA GENETICS on the Hamilton's STARlet platform. We present the comparison of results between this automated approach, used for more than 1,000 DNA patients' samples, and the performances of the manual protocol evaluated by SOPHiA GENETICS onto 240 samples summarized in their HCS evaluation study.Results: We demonstrate that this automated workflow achieved the same expected goals of manual setup in terms of coverages and reads uniformity, with extremely lower standard deviations among samples considering the sequencing reads mapped onto the regions of interest.Conclusions: This automated solution offers same reliable and affordable NGS data, but with the essential advantages of a flexible, automated and integrated framework, minimizing possible human errors and depicting a laboratory's walk-away scenario.
2021
- Automation of a capture-based NGS workflow: one thousand patients experience in a diagnostic clinical routine framework
[Abstract in Rivista]
Tenedini, E.; Celestini, F.; Iapicca, P.; Marino, M.; Castellano, S.; Artuso, L.; Biagiarelli, F.; Cortesi, L.; Toss, A.; Barbieri, E.; Roncucci, L.; Pedroni, M.; Manfredini, R.; Luppi, M.; Trenti, T.; Tagliafico, E.
abstract
2021
- Characterization of new ATM deletion associated with hereditary breast cancer
[Articolo su rivista]
Parenti, S.; Rabacchi, C.; Marino, M.; Tenedini, E.; Artuso, L.; Castellano, S.; Carretta, C.; Mallia, S.; Cortesi, L.; Toss, A.; Barbieri, E.; Manfredini, R.; Luppi, M.; Trenti, T.; Tagliafico, E.
abstract
Next-generation sequencing (NGS)-based cancer risk screening with multigene panels has become the most successful method for programming cancer prevention strategies. ATM germ-line heterozygosity has been described to increase tumor susceptibility. In particular, families carrying heterozygous germ-line variants of ATM gene have a 5-to 9-fold risk of developing breast cancer. Recent studies identified ATM as the second most mutated gene after CHEK2 in BRCA-negative patients. Nowadays, more than 170 missense variants and several truncating mutations have been identified in ATM gene. Here, we present the molecular characterization of a new ATM deletion, identified thanks to the CNV algorithm implemented in the NGS analysis pipeline. An automated workflow implementing the SOPHiA Genetics’ Hereditary Cancer Solution (HCS) protocol was used to generate NGS libraries that were sequenced on Illumina MiSeq Platform. NGS data analysis allowed us to identify a new inactivating deletion of exons 19–27 of ATM gene. The deletion was characterized both at the DNA and RNA level.
2021
- Gene expression profile correlates with molecular and clinical features in patients with myelofibrosis
[Articolo su rivista]
Rontauroli, S.; Castellano, S.; Guglielmelli, P.; Zini, R.; Bianchi, E.; Genovese, E.; Carretta, C.; Parenti, S.; Fantini, S.; Mallia, S.; Tavernari, L.; Sartini, S.; Mirabile, M.; Mannarelli, C.; Gesullo, F.; Pacilli, A.; Pietra, D.; Rumi, E.; Salmoiraghi, S.; Mora, B.; Villani, L.; Grilli, A.; Rosti, V.; Barosi, G.; Passamonti, F.; Rambaldi, A.; Malcovati, L.; Cazzola, M.; Bicciato, S.; Tagliafico, E.; Vannucchi, A. M.; Manfredini, R.
abstract
Myelofibrosis (MF) belongs to the family of classic Philadelphia-negative myeloproliferative neoplasms (MPNs). It can be primary myelofibrosis (PMF) or secondary myelofibrosis (SMF) evolving from polycythemia vera (PV) or essential thrombocythemia (ET). Despite the differences, PMF and SMF patients are currently managed in the same way, and prediction of survival is based on the same clinical and genetic features. In the last few years, interest has grown concerning the ability of gene expression profiles (GEPs) to provide valuable prognostic information. Here, we studied the GEPs of granulocytes from 114 patients with MF, using a microarray platform to identify correlations with patient characteristics and outcomes. Cox regression analysis led to the identification of 201 survival-related transcripts characterizing patients who are at high risk for death. High-risk patients identified by this gene signature displayed an inferior overall survival and leukemia-free survival, together with clinical and molecular detrimental features included in contemporary prognostic models, such as the presence of high molecular risk mutations. The high-risk group was enriched in post-PV and post-ET MF and JAK2V617F homozygous patients, whereas pre-PMF was more frequent in the low-risk group. These results demonstrate that GEPs in MF patients correlate with their molecular and clinical features, particularly their survival, and represent the proof of concept that GEPs might provide complementary prognostic information to be applied in clinical decision making.
2021
- Increased Plasma Levels of lncRNAs LINC01268, GAS5 and MALAT1 Correlate with Negative Prognostic Factors in Myelofibrosis
[Articolo su rivista]
Fantini, Sebastian; Rontauroli, Sebastiano; Sartini, Stefano; Mirabile, Margherita; Bianchi, Elisa; Badii, Filippo; Maccaferri, Monica; Guglielmelli, Paola; Ottone, Tiziana; Palmieri, Raffaele; Genovese, Elena; Carretta, Chiara; Parenti, Sandra; Mallia, Selene; Tavernari, Lara; Salvadori, Costanza; Gesullo, Francesca; Maccari, Chiara; Zizza, Michela; Grande, Alexis; Salmoiraghi, Silvia; Mora, Barbara; Potenza, Leonardo; Rosti, Vittorio; Passamonti, Francesco; Rambaldi, Alessandro; Voso, Maria Teresa; Mecucci, Cristina; Tagliafico, Enrico; Luppi, Mario; Vannucchi, Alessandro Maria; Manfredini, Rossella
abstract
: Long non-coding RNAs (lncRNAs) have been recently described as key mediators in the development of hematological malignancies. In the last years, circulating lncRNAs have been proposed as a new class of non-invasive biomarkers for cancer diagnosis and prognosis and to predict treatment response. The present study is aimed to investigate the potential of circulating lncRNAs as non-invasive prognostic biomarkers in myelofibrosis (MF), the most severe among Philadelphia-negative myeloproliferative neoplasms. We detected increased levels of seven circulating lncRNAs in plasma samples of MF patients (n = 143), compared to healthy controls (n = 65). Among these, high levels of LINC01268, MALAT1 or GAS5 correlate with detrimental clinical variables, such as high count of leukocytes and CD34+ cells, severe grade of bone marrow fibrosis and presence of splenomegaly. Strikingly, high plasma levels of LINC01268 (p = 0.0018), GAS5 (p = 0.0008) or MALAT1 (p = 0.0348) are also associated with a poor overall-survival while high levels of LINC01268 correlate with a shorter leukemia-free-survival. Finally, multivariate analysis demonstrated that the plasma level of LINC01268 is an independent prognostic variable, suggesting that, if confirmed in future in an independent patients' cohort, it could be used for further studies to design an updated classification model for MF patients.
2021
- Inflammatory microenvironment and specific t cells in myeloproliferative neoplasms: Immunopathogenesis and novel immunotherapies
[Articolo su rivista]
Nasillo, V.; Riva, G.; Paolini, A.; Forghieri, F.; Roncati, L.; Lusenti, B.; Maccaferri, M.; Messerotti, A.; Pioli, V.; Gilioli, A.; Bettelli, F.; Giusti, D.; Barozzi, P.; Lagreca, I.; Maffei, R.; Marasca, R.; Potenza, L.; Comoli, P.; Manfredini, R.; Maiorana, A.; Tagliafico, E.; Luppi, M.; Trenti, T.
abstract
The Philadelphia-negative myeloproliferative neoplasms (MPNs) are malignancies of the hematopoietic stem cell (HSC) arising as a consequence of clonal proliferation driven by somatically acquired driver mutations in discrete genes (JAK2, CALR, MPL). In recent years, along with the advances in molecular characterization, the role of immune dysregulation has been achieving increasing relevance in the pathogenesis and evolution of MPNs. In particular, a growing number of studies have shown that MPNs are often associated with detrimental cytokine milieu, expansion of the monocyte/macrophage compartment and myeloid-derived suppressor cells, as well as altered functions of T cells, dendritic cells and NK cells. Moreover, akin to solid tumors and other hemato-logical malignancies, MPNs are able to evade T cell immune surveillance by engaging the PD-1/PD-L1 axis, whose pharmacological blockade with checkpoint inhibitors can successfully restore effective antitumor responses. A further interesting cue is provided by the recent discovery of the high immunogenic potential of JAK2V617F and CALR exon 9 mutations, that could be harnessed as in-triguing targets for innovative adoptive immunotherapies. This review focuses on the recent insights in the immunological dysfunctions contributing to the pathogenesis of MPNs and outlines the potential impact of related immunotherapeutic approaches.
2021
- Ivar, an interpretation‐oriented tool to manage the update and revision of variant annotation and classification
[Articolo su rivista]
Castellano, S.; Cestari, F.; Faglioni, G.; Tenedini, E.; Marino, M.; Artuso, L.; Manfredini, R.; Luppi, M.; Trenti, T.; Tagliafico, E.
abstract
The rapid evolution of Next Generation Sequencing in clinical settings, and the resulting challenge of variant reinterpretation given the constantly updated information, require robust data management systems and organized approaches. In this paper, we present iVar: a freely available and highly customizable tool with a user‐friendly web interface. It represents a platform for the unified management of variants identified by different sequencing technologies. iVar accepts variant call format (VCF) files and text annotation files and elaborates them, optimizing data organization and avoiding redundancies. Updated annotations can be periodically re‐uploaded and associated with variants as historically tracked attributes, i.e., modifications can be recorded whenever an updated value is imported, thus keeping track of all changes. Data can be visualized through variant‐centered and sample‐centered interfaces. A customizable search function can be exploited to periodically check if pathogenicity‐related data of a variant has changed over time. Patient recontacting ensuing from variant reinterpretation is made easier by iVar through the effective identification of all patients present in the database carrying a specific variant. We tested iVar by uploading 4171 VCF files and 1463 annotation files, obtaining a database of 4166 samples and 22,569 unique variants. iVar has proven to be a useful tool with good performance in terms of collecting and managing data from a medium‐throughput laboratory.
2021
- Magnesium favors the capacity of vitamin d3 to induce the monocyte differentiation of u937 cells
[Articolo su rivista]
Parenti, S.; Sandoni, L.; Montanari, M.; Zanocco-Marani, T.; Anesi, A.; Iotti, S.; Manfredini, R.; Frassineti, C.; Davalli, P.; Grande, A.
abstract
The hematopoietic U937 cells are able to differentiate into monocytes, macrophages, or osteoclasts when stimulated, respectively, with vitamin D3 (VD3), phorbol 12-myristate 13-acetate (PMA) or PMA plus VD3. We have previously demonstrated that magnesium (Mg) strongly potentiates the osteoclastic differentiation of U937 cells. In this study, we investigated whether such an effect may be ascribed to a capacity of Mg to modulate the monocyte differentiation of U937 cells and/or to an ability of Mg and VD3 to act directly and independently on the early phases of the osteoclastic differentiation. To address this issue, we subjected U937 cells to an individual and combined treatment with Mg and VD3 and then we analyzed, by flow cytometry and quantitative real-time polymerase chain reaction, the expression of a number of genes related to the early phases of the differentiation pathways under consideration. The results obtained indicated that Mg favors the monocyte differentiation of U937 cells induced by VD3 and at the same time, Mg contrasts the inhibitory effect that VD3 exerts on the osteoclastic differentiation in the absence of PMA. The crucial and articulated role played by Mg in diverse pathways of the osteoclastic differentiation of U973 cells is emphasized.
2021
- Monocyte Distribution Width (MDW) as novel inflammatory marker with prognostic significance in COVID-19 patients
[Articolo su rivista]
Riva, G.; Castellano, S.; Nasillo, V.; Ottomano, A. M.; Bergonzini, G.; Paolini, A.; Lusenti, B.; Milic, J.; De Biasi, S.; Gibellini, L.; Cossarizza, A.; Busani, S.; Girardis, M.; Guaraldi, G.; Mussini, C.; Manfredini, R.; Luppi, M.; Tagliafico, E.; Trenti, T.
abstract
Monocyte Distribution Width (MDW), a new cytometric parameter correlating with cytomorphologic changes occurring upon massive monocyte activation, has recently emerged as promising early biomarker of sepsis. Similar to sepsis, monocyte/macrophage subsets are considered key mediators of the life-threatening hyper-inflammatory disorder characterizing severe COVID-19. In this study, we longitudinally analyzed MDW values in a cohort of 87 COVID-19 patients consecutively admitted to our hospital, showing significant correlations between MDW and common inflammatory markers, namely CRP (p < 0.001), fibrinogen (p < 0.001) and ferritin (p < 0.01). Moreover, high MDW values resulted to be prognostically associated with fatal outcome in COVID-19 patients (AUC = 0.76, 95% CI: 0.66–0.87, sensitivity 0.75, specificity 0.70, MDW threshold 26.4; RR = 4.91, 95% CI: 1.73–13.96; OR = 7.14, 95% CI: 2.06–24.71). This pilot study shows that MDW can be useful in the monitoring of COVID-19 patients, as this innovative hematologic biomarker is: (1) easy to obtain, (2) directly related to the activation state of a fundamental inflammatory cell subset (i.e. monocytes, pivotal in both cytokine storm and sepsis immunopathogenesis), (3) well correlated with clinical severity of COVID-19-associated inflammatory disorder, and, in turn, (4) endowed with relevant prognostic significance. Additional studies are needed to define further the clinical impact of MDW testing in the management of COVID-19 patients.
2021
- Multiparametric flow cytometry for MRD monitoring in hematologic malignancies: Clinical applications and new challenges
[Articolo su rivista]
Riva, G.; Nasillo, V.; Ottomano, A. M.; Bergonzini, G.; Paolini, A.; Forghieri, F.; Lusenti, B.; Barozzi, P.; Lagreca, I.; Fiorcari, S.; Martinelli, S.; Maffei, R.; Marasca, R.; Potenza, L.; Comoli, P.; Manfredini, R.; Tagliafico, E.; Trenti, T.; Luppi, M.
abstract
In hematologic cancers, Minimal Residual Disease (MRD) monitoring, using either molecular (PCR) or immunophenotypic (MFC) diagnostics, allows the identification of rare cancer cells, readily detectable either in the bone marrow or in the peripheral blood at very low levels, far below the limit of classic microscopy. In this paper, we outlined the state-of-the-art of MFC-based MRD detection in different hematologic settings, highlighting main recommendations and new challenges for using such a method in patients with acute leukemias or chronic hematologic neoplasms. The combination of new molecular technologies with advanced flow cytometry is progressively allowing clinicians to design a personalized therapeutic path, proportionate to the biological aggressiveness of the disease, in particular by using novel immunotherapies, in view of a modern decision-making process, based on precision medicine. Along with the evolution of immunophenotypic and molecular diagnostics, the assessment of Minimal Residual Disease (MRD) has progressively become a keystone in the clinical management of hematologic malignancies, enabling valuable post-therapy risk stratifications and guiding risk-adapted therapeutic approaches. However, specific prognostic values of MRD in different hematological settings, as well as its appropriate clinical uses (basically, when to measure it and how to deal with different MRD levels), still need further investigations, aiming to improve standardization and harmonization of MRD monitoring protocols and MRD-driven therapeutic strategies. Currently, MRD measurement in hematological neoplasms with bone marrow involvement is based on advanced highly sensitive methods, able to detect either specific genetic abnormalities (by PCRbased techniques and next-generation sequencing) or tumor-associated immunophenotypic profiles (by multiparametric flow cytometry, MFC). In this review, we focus on the growing clinical role for MFC-MRD diagnostics in hematological malignancies-from acute myeloid and lymphoblastic leukemias (AML, B-ALL and T-ALL), to chronic lymphocytic leukemia (CLL) and multiple myeloma (MM)-providing a comparative overview on technical aspects, clinical implications, advantages and pitfalls of MFC-MRD monitoring in different clinical settings.
2021
- Mutated clones driving leukemic transformation are already detectable at the single-cell level in CD34-positive cells in the chronic phase of primary myelofibrosis
[Articolo su rivista]
Parenti, Sandra; Rontauroli, Sebastiano; Carretta, Chiara; Mallia, Selene; Genovese, Elena; Chiereghin, Chiara; Peano, Clelia; Tavernari, Lara; Bianchi, Elisa; Fantini, Sebastian; Sartini, Stefano; Romano, Oriana; Bicciato, Silvio; Tagliafico, Enrico; Della Porta, Matteo; Manfredini, Rossella
abstract
Disease progression of myeloproliferative neoplasms is the result of increased genomic complexity. Since the ability to predict disease evolution is crucial for clinical decisions, we studied single-cell genomics and transcriptomics of CD34-positive cells from a primary myelofibrosis (PMF) patient who progressed to acute myeloid leukemia (AML) while receiving Ruxolitinib. Single-cell genomics allowed the reconstruction of clonal hierarchy and demonstrated that TET2 was the first mutated gene while FLT3 was the last one. Disease evolution was accompanied by increased clonal heterogeneity and mutational rate, but clones carrying TP53 and FLT3 mutations were already present in the chronic phase. Single-cell transcriptomics unraveled repression of interferon signaling suggesting an immunosuppressive effect exerted by Ruxolitinib. Moreover, AML transformation was associated with a differentiative block and immune escape. These results suggest that single-cell analysis can unmask tumor heterogeneity and provide meaningful insights about PMF progression that might guide personalized therapy.
2021
- Neoantigen-specific T-cell immune responses: The paradigm of NPM1-mutated acute myeloid leukemia
[Articolo su rivista]
Forghieri, F.; Riva, G.; Lagreca, I.; Barozzi, P.; Bettelli, F.; Paolini, A.; Nasillo, V.; Lusenti, B.; Pioli, V.; Giusti, D.; Gilioli, A.; Colasante, C.; Galassi, L.; Catellani, H.; Donatelli, F.; Talami, A.; Maffei, R.; Martinelli, S.; Potenza, L.; Marasca, R.; Tagliafico, E.; Manfredini, R.; Trenti, T.; Comoli, P.; Luppi, M.
abstract
The C-terminal aminoacidic sequence from NPM1-mutated protein, absent in normal human tissues, may serve as a leukemia-specific antigen and can be considered an ideal target for NPM1-mutated acute myeloid leukemia (AML) immunotherapy. Different in silico instruments and in vitro/ex vivo immunological platforms have identified the most immunogenic epitopes from NPM1-mutated protein. Spontaneous development of endogenous NPM1-mutated-specific cytotoxic T cells has been observed in patients, potentially contributing to remission maintenance and prolonged survival. Genetically engineered T cells, namely CAR-T or TCR-transduced T cells, directed against NPM1-mutated peptides bound to HLA could prospectively represent a promising therapeutic approach. Although either adoptive or vaccine-based immunotherapies are unlikely to be highly effective in patients with full-blown leukemia, these strategies, potentially in combination with immune-checkpoint inhibitors, could be promising in maintaining remission or preemptively eradicat-ing persistent measurable residual disease, mainly in patients ineligible for allogeneic hematopoietic stem cell transplant (HSCT). Alternatively, neoantigen-specific donor lymphocyte infusion derived from healthy donors and targeting NPM1-mutated protein to selectively elicit graft-versus-leukemia effect may represent an attractive option in subjects experiencing post-HSCT relapse. Future studies are warranted to further investigate dynamics of NPM1-mutated-specific immunity and explore whether novel individualized immunotherapies may have potential clinical utility in NPM1-mutated AML patients.
2021
- Pre-existing cytopenia heralding de novo acute myeloid leukemia: Uncommon presentation of NPM1-mutated AML in a single-center study
[Articolo su rivista]
Galassi, L.; Colasante, C.; Bettelli, F.; Gilioli, A.; Pioli, V.; Giusti, D.; Morselli, M.; Paolini, A.; Nasillo, V.; Lusenti, B.; Colaci, E.; Donatelli, F.; Catellani, H.; Pozzi, S.; Barbieri, E.; del Rosso, M. N.; Barozzi, P.; Lagreca, I.; Martinelli, S.; Maffei, R.; Riva, G.; Tenedini, E.; Roncati, L.; Marasca, R.; Potenza, L.; Comoli, P.; Trenti, T.; Manfredini, R.; Tagliafico, E.; Luppi, M.; Forghieri, F.
abstract
2020
- Genomic analysis of hematopoietic stem cell at the single-cell level: Optimization of cell fixation and whole genome amplification (WGA) protocol
[Articolo su rivista]
Carretta, C.; Mallia, S.; Genovese, E.; Parenti, S.; Rontauroli, S.; Bianchi, E.; Fantini, S.; Sartini, S.; Tavernari, L.; Tagliafico, E.; Manfredini, R.
abstract
Single-cell genomics has become the method of choice for the study of heterogeneous cell populations and represents an elective application in defining the architecture and clonal evolution in hematological neoplasms. Reconstructing the clonal evolution of a neoplastic population therefore represents the main way to understand more deeply the pathogenesis of the neoplasm, but it is also a potential tool to understand the evolution of the tumor population with respect to its response to therapy. Pre-analytical phase for single-cell genomics analysis is crucial to obtain a cell population suitable for single-cell sorting, and whole genome amplification is required to obtain the necessary amount of DNA from a single cell in order to proceed with sequencing. Here, we evaluated the impact of different methods of cellular immunostaining, fixation and whole genome amplification on the efficiency and yield of single-cell sequencing.
2020
- Preclinical study for treatment of hypospadias by advanced therapy medicinal products
[Articolo su rivista]
Sceberras, V.; Attico, E.; Bianchi, E.; Galaverni, G.; Melonari, M.; Corradini, F.; Fantacci, M.; Ribbene, A.; Losi, L.; Balò, S.; Lazzeri, M.; Trombetta, C.; Rizzo, M.; Manfredini, R.; Barbagli, G.; Pellegrini, G.
abstract
Purpose
This paper explores the feasibility of a new therapy for the treatment of hypospadias patients. Hypospadias is a very common congenital malformation of male genitals, with very high rate of recurrences after surgery. The field of regenerative medicine, which offers innovative solutions for many pathologies, still does not offer reliable solution for this pathology. Here, we propose quality, safety, and clinical feasibility assessment for an oral mucosa advanced therapy medicinal product (ATMP) grown on a biocompatible scaffold for a clinical study on urethral reconstruction of hypospadias patients.
Methods
Urethral and oral mucosal epithelia from donor biopsies were cultivated between two fibrin layers, under clinical-grade conditions for cell and tissue characterization and comparison, aimed at tissue engineering. In addition, single-clone analyses were performed to analyze gene expression profiles of the two epithelia by microarray technology.
Results
Oral mucosa appeared suitable for urethral reconstruction. The resulting ATMP was proven to maintain stem cells and regenerative potency. The preclinical safety studies were performed on human tissues to assess abnormalities and tumorigenicity, and confirmed the safety of the ATMP. Finally, the patient selection and the clinical protocol for the upcoming clinical trial were defined.
Conclusions
Against this backdrop, in this paper, we are proposing a new reproducible and reliable ATMP for the treatment of hypospadias.
2020
- Wnt/CTNNB1 signal transduction pathway inhibits the expression of ZFP36 in squamous cell carcinoma, by inducing transcriptional repressors SNAI1, SLUG and TWIST
[Articolo su rivista]
Zanfi, E. D.; Fantini, S.; Lotti, R.; Bertesi, M.; Marconi, A.; Grande, A.; Manfredini, R.; Pincelli, C.; Zanocco-Marani, T.
abstract
The Wnt/CTNNB1 pathway is often deregulated in epithelial tumors. The ZFP36 gene, encoding the mRNA binding protein Tristetraprolin (TTP), is downregulated in several cancers, where it has been described to behave as a tumor suppressor. By this report, we show that Wnt/CTNNB1 pathway is constitutively activated, and ZFP36 expression is downregulated in Squamous Cell Carcinoma (SCC) cell lines compared to normal keratinocytes. Moreover, we suggest that the decrease of ZFP36 expression might depend on the activity of transcriptional repressors SNAI1, SLUG and TWIST, whose expression is induced by Wnt/CTNNB1, highlighting a potential regulatory mechanism underlying ZFP36 downregulation in epithelial cancers.
2019
- Calreticulin Ins5 and Del52 mutations impair unfolded protein and oxidative stress responses in K562 cells expressing CALR mutants
[Articolo su rivista]
Salati, Simona; Genovese, Elena; Carretta, Chiara; Zini, Roberta; Bartalucci, Niccolò; Prudente, Zelia; Pennucci, Valentina; Ruberti, Samantha; Rossi, Chiara; Rontauroli, Sebastiano; Enzo, Elena; Calabresi, Laura; Balliu, Manjola; Mannarelli, Carmela; Bianchi, Elisa; Guglielmelli, Paola; Tagliafico, Enrico; Vannucchi, Alessandro M; Manfredini, Rossella
abstract
Somatic mutations of calreticulin (CALR) have been described in approximately 60-80% of JAK2 and MPL unmutated Essential Thrombocythemia and Primary Myelofibrosis patients. CALR is an endoplasmic reticulum (ER) chaperone responsible for proper protein folding and calcium retention. Recent data demonstrated that the TPO receptor (MPL) is essential for the development of CALR mutant-driven Myeloproliferative Neoplasms (MPNs). However, the precise mechanism of action of CALR mutants haven't been fully unraveled. In this study, we showed that CALR mutants impair the ability to respond to the ER stress and reduce the activation of the pro-apoptotic pathway of the unfolded protein response (UPR). Moreover, our data demonstrated that CALR mutations induce increased sensitivity to oxidative stress, leading to increase oxidative DNA damage. We finally demonstrated that the downmodulation of OXR1 in CALR-mutated cells could be one of the molecular mechanisms responsible for the increased sensitivity to oxidative stress mediated by mutant CALR. Altogether, our data identify novel mechanisms collaborating with MPL activation in CALR-mediated cellular transformation. CALR mutants negatively impact on the capability of cells to respond to oxidative stress leading to genomic instability and on the ability to react to ER stress, causing resistance to UPR-induced apoptosis.
2019
- Spectrum of ASXL1 mutations in primary myelofibrosis: Prognostic impact of the ASXL1 p.G646Wfs∗12 mutation
[Articolo su rivista]
Rotunno, G.; Mannarelli, C.; Brogi, G.; Pacilli, A.; Gesullo, F.; Mannelli, F.; Fiaccabrino, S.; Sordi, B.; Paoli, C.; Marone, I.; Rumi, E.; Manfredini, R.; Barosi, G.; Cazzola, M.; Vannucchi, A. M.; Guglielmelli, P.
abstract
N/A
2019
- miRNA142-3p targets Tet2 and impairs Treg differentiation and stability in models of type 1 diabetes
[Articolo su rivista]
Scherm, M. G.; Serr, I.; Zahm, A. M.; Schug, J.; Bellusci, S.; Manfredini, R.; Salb, V. K.; Gerlach, K.; Weigmann, B.; Ziegler, A. -G.; Kaestner, K. H.; Daniel, C.
abstract
In type 1 diabetes, the appearance of islet autoantibodies indicates the onset of islet autoimmunity, often many years before clinical symptoms arise. While T cells play a major role in the destruction of pancreatic beta cells, molecular underpinnings promoting aberrant T cell activation remain poorly understood. Here, we show that during islet autoimmunity an miR142-3p/Tet2/Foxp3 axis interferes with the efficient induction of regulatory T (Treg) cells, resulting in impaired Treg stability in mouse and human. Specifically, we demonstrate that miR142-3p is induced in islet autoimmunity and that its inhibition enhances Treg induction and stability, leading to reduced islet autoimmunity in non-obese diabetic mice. Using various cellular and molecular approaches we identify Tet2 as a direct target of miR142-3p, thereby linking high miR142-3p levels to epigenetic remodeling in Tregs. These findings offer a mechanistic model where during islet autoimmunity miR142-3p/Tet2-mediated Treg instability contributes to autoimmune activation and progression.
2018
- Bone marrow-specific loss of ABI1 induces myeloproliferative neoplasm with features resembling, human myelofibrosis
[Articolo su rivista]
Chorzalska, Anna; Morgan, John; Ahsan, Nagib; Treaba, Diana O.; Olszewski, Adam J.; Petersen, Max; Kingston, Nathan; Cheng, Yan; Lombardo, Kara; Schorl, Christoph; Yu, Xiaoqing; Zini, Roberta; Pacilli, Annalisa; Tepper, Alexander; Coburn, Jillian; Hryniewicz-Jankowska, Anita; Zhao, Ting C.; Oancea, Elena; Reagan, John L.; Liang, Olin; Kotula, Leszek; Quesenberry, Peter J.; Gruppuso, Philip A.; Manfredini, Rossella; Vannucchi, Alessandro Maria; Dubielecka, Patrycja M.
abstract
Although the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs) is linked to constitutive activation of the JAK-STAT pathway, JAK inhibitors have neither curative nor MPN-stem cell-eradicating potential, indicating that other targetable mechanisms are contributing to the pathophysiology of MPNs. We previously demonstrated that Abelson interactor 1 (Abi-1), a negative regulator of Abelson kinase 1, functions as a tumor suppressor. Here we present data showing that bone marrow-specific deletion of Abi1 in a novel mouse model leads to development of an MPNlike phenotype resembling human PMF. Abi1 loss resulted in a significant increase in the activity of the Src family kinases (SFKs), STAT3, and NF-κB signaling. We also observed impairment of hematopoietic stem cell self-renewal and fitness, as evidenced in noncompetitive and competitive bone marrow transplant experiments. CD34 + hematopoietic progenitors and granulocytes from patients with PMF showed decreased levels of ABI1 transcript as well as increased activity of SFKs, STAT3, and NF-κB. In aggregate, our data link the loss of Abi-1 function to hyperactive SFKs/STAT3/NF-κB signaling and suggest that this signaling axis may represent a regulatory module involved in the molecular pathophysiology of PMF.
2018
- Calreticulin affects hematopoietic stem/progenitor cell fate by impacting erythroid and megakaryocytic differentiation
[Articolo su rivista]
Salati, Simona; Prudente, Zelia; Genovese, Elena; Pennucci, Valentina; Rontauroli, Sebastiano; Bartalucci, Niccolo'; Mannarelli, Carmela; Ruberti, Samantha; Zini, Roberta; Rossi, Chiara; Bianchi, Elisa; Guglielmelli, Paola; Tagliafico, Enrico; Vannucchi, Alessandro M; Manfredini, Rossella
abstract
Calreticulin (CALR) is a chaperone protein that localizes primarily to the endoplasmic reticulum (ER) lumen where it is responsible for the control of proper folding of neo-synthesized glycoproteins and for the retention of calcium. Recently, mutations affecting exon 9 of the CALR gene have been described in approximately 40% of patients with myeloproliferative neoplasms (MPNs). Although the role of mutated CALR in the development of MPNs has begun to be clarified, there are still no data available on the function of wild-type (WT) CALR during physiological hematopoiesis. In order to shed light on the role of WT CALR during normal hematopoiesis, we performed gene silencing and overexpression experiments in Hematopoietic Stem Progenitor Cells (HSPCs). Our results showed that CALR overexpression is able to affect physiological hematopoiesis by enhancing both erythroid and megakaryocytic (MK) differentiation. In agreement with overexpression data, CALR silencing caused a significant decrease in both erythroid and MK differentiation of human HSPCs. Gene expression profiling (GEP) analysis showed that CALR is able to affect the expression of several genes involved in HSPCs differentiation towards both the erythroid and MK lineages. Moreover, GEP data also highlighted the modulation of several genes involved in ER stress response, unfolded protein response (UPR), DNA repair and of several genes already described to play a role in MPN development, such as pro-inflammatory cytokines and hematological neoplasms-related markers. Altogether, our data unraveled a new and unexpected role for CALR in the regulation of normal hematopoietic differentiation. Moreover, by showing the impact of CALR on the expression of genes involved in several biological processes already described in cellular transformation, our data strongly suggest a more complex role for CALR in MPN development that goes beyond the activation of the THPO receptor and involves ER stress response, UPR and DNA repair.
2018
- Cytokine-induced killer cells express CD39, CD38, CD203a, CD73 ectoenzymes and P1 adenosinergic receptors
[Articolo su rivista]
Horenstein, Alberto L.; Chillemi, Antonella; Zini, Roberta; Quarona, Valeria; Bianchi, Nicoletta; Manfredini, Rossella; Gambari, Roberto; Malavasi, Fabio; Ferrari, Davide
abstract
Cytokine-induced killer (CIK) cells, a heterogeneous T cell population obtained by in vitro differentiation of peripheral blood mononuclear cells (PBMC), represent a promising immunological approach in cancer. Numerous studies have explored the role of CD38, CD39, CD203a/PC-1, and CD73 in generating extracellular adenosine (ADO) and thus in shaping the tumor niche in favor of proliferation. The findings shown here reveal that CIK cells are able to produce extracellular ADO via traditional (CD39/CD73) and/or alternative (CD38/CD203a/CD73 or CD203a/CD73) pathways. Transcriptome analysis showed the mRNA expression of these molecules and their modulation during PBMC to CIK differentiation. When PBMC from normal subjects or cancer bearing patients were differentiated into CIK cells under normoxic conditions, CD38 and CD39 were greatly up-regulated while the number of CD203a, and CD73 positive cells underwent minor changes. Since hypoxic conditions are often found in tumors, we asked whether CD39, CD38, CD203a, and CD73 expressed by CIK cells were modulated by hypoxia. PBMC isolated from cancer patients and differentiated into CIK cells in hypoxic conditions did not show relevant changes in CD38, CD39, CD73, CD203a, and CD26. CIK cells also expressed A1, A2A, and A2B ADO receptors and they only underwent minor changes as a consequence of hypoxia. The present study sheds light on a previously unknown functional aspect of CIK cells, opening the possibility of pharmacologically modulated ADO-generating ectoezymes to improve CIK cells performance.
2018
- Differential proteomic profile of leukemic CD34+ progenitor cells from chronic myeloid leukemia patients
[Articolo su rivista]
Ricciardi, Maria Rosaria; Salvestrini, Valentina; Licchetta, Roberto; Mirabilii, Simone; Forcato, Mattia; Gugliotta, Gabriele; Salati, Simona; Castagnetti, Fausto; Rosti, Gianantonio; Breccia, Massimo; Alimena, Giuliana; Manfredini, Rossella; Bicciato, Silvio; Lemoli, Roberto Massimo; Tafuri, Agostino
abstract
Chronic Myeloid Leukemia (CML) is a stem cell disease sustained by a rare population of quiescent cells which are to some extent resistant to tyrosine kinase inhibitors (TKIs). BCR-ABL oncogene activates multiple cross-talking signal transduction pathways (STP), such as RAS/MEK/ERK, PI3K/Akt, Wnt and STAT5, contributing to abnormal proliferation of clonal cells. From this perspective, the aim of this study was to analyze the expression and activation profile of STP involved in the mechanisms of cell proliferation/quiescence and survival of the progenitor CD34+ cells from chronic phase (CP) CML. Our results showed that CP-CML CD34+ progenitors were characterized by significant lower phosphorylation of proteins involved in the regulation of growth and cell survival, such as tyrosine kinases of the Src family and members of STAT family, and by a significant higher phosphorylation of p53 (Ser15), compared to normal CD34+ cells from healthy donors. Consistent with these results, cell cycle analysis demonstrated that CP-CML CD34+ cells were characterized by higher percentage of cells in G0-phase compared to normal CD34+ cells. Analysis of expression profile on proteins involved in the apoptotic machinery revealed that, in addition, CD34+ cells from CP-CML were characterized by a significant lower expression of catalase and higher expression of HSP27 and FADD. In sum, we report that CD34+ cells from CP-CML are characterized by a proteomic and phospho-proteomic profile that promotes quiescence through the inhibition of proliferation and the promotion of survival. This differential signaling activation network may be addressed by novel targeted therapies aimed at eradicating CML stem cells.
2018
- Involvement of MAF/SPP1 axis in the development of bone marrow fibrosis in PMF patients
[Articolo su rivista]
Ruberti, S; Bianchi, E; Guglielmelli, P; Rontauroli, S; Barbieri, G; Tavernari, L; Fanelli, T; Norfo, R; Pennucci, V; Fattori, G. Corbizi; Mannarelli, C; Bartalucci, N; Mora, B; Elli, L; Avanzini, M. A; Rossi, C; Salmoiraghi, S; Zini, R; Salati, S; Prudente, Z; Rosti, V; Passamonti, F; Rambaldi, A; Ferrari, S; Tagliafico, E; Vannucchi, A. M; Manfredini, R.
abstract
Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. We recently described the upregulation of MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) in PMF CD34+ hematopoietic progenitor cells (HPCs) compared to healthy donor. Here we demonstrated that MAF is also upregulated in PMF compared with the essential thrombocytemia (ET) and polycytemia vera (PV) HPCs. MAF overexpression and knockdown experiments shed some light into the role of MAF in PMF pathogenesis, by demonstrating that MAF favors the megakaryocyte and monocyte/macrophage commitment of HPCs and leads to the increased expression of proinflammatory and profibrotic mediators. Among them, we focused our further studies on SPP1 and LGALS3. We assessed SPP1 and LGALS3 protein levels in 115 PMF, 47 ET and 24 PV patients plasma samples and we found that SPP1 plasma levels are significantly higher in PMF compared with ET and PV patients. Furthermore, in vitro assays demonstrated that SPP1 promotes fibroblasts and mesenchymal stromal cells proliferation and collagen production. Strikingly, clinical correlation analyses uncovered that higher SPP1 plasma levels in PMF patients correlate with a more severe fibrosis degree and a shorter overall survival. Collectively our data unveil that MAF overexpression contributes to PMF pathogenesis by driving the deranged production of the profibrotic mediator SPP1.
2018
- Role of TGF-β1/miR-382-5p/SOD2 axis in the induction of oxidative stress in CD34+ cells from primary myelofibrosis
[Articolo su rivista]
Rossi, Chiara; Zini, Roberta; Rontauroli, Sebastiano; Ruberti, Samantha; Prudente, Zelia; Barbieri, Greta; Bianchi, Elisa; Salati, Simona; Genovese, Elena; Bartalucci, Niccolò; Guglielmelli, Paola; Tagliafico, Enrico; Rosti, Vittorio; Barosi, Giovanni; Vannucchi, Alessandro M.; Manfredini, Rossella
abstract
Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by an excessive production of pro-inflammatory cytokines resulting in chronic inflammation and genomic instability. Besides the driver mutations in JAK2, MPL, and CALR genes, the deregulation of miRNA expression may also contribute to the pathogenesis of PMF. To this end, we recently reported the upregulation of miR-382-5p in PMF CD34+ cells. In order to unveil the mechanistic details of the role of miR-382-5p in pathogenesis of PMF, we performed gene expression profiling of CD34+ cells overexpressing miR-382-5p. Among the downregulated genes, we identified superoxide dismutase 2 (SOD2), which is a predicted target of miR-382-5p. Subsequently, we confirmed miR-382-5p/SOD2 interaction by luciferase assay and we showed that miR-382-5p overexpression in CD34+ cells causes the decrease in SOD2 activity leading to reactive oxygen species (ROS) accumulation and oxidative DNA damage. In addition, our data indicate that inhibition of miR-382-5p in PMF CD34+ cells restores SOD2 function, induces ROS disposal, and reduces DNA oxidation. Since the pro-inflammatory cytokine transforming growth factor-β1 (TGF-β1) is a key player in PMF pathogenesis, we further investigated the effect of TGF-β1 on ROS and miR-382-5p levels. Our data showed that TGF-β1 treatment enhances miR-382-5p expression and reduces SOD2 activity leading to ROS accumulation. Finally, inhibition of TGF-β1 signaling in PMF CD34+ cells by galunisertib significantly reduced miR-382-5p expression and ROS accumulation and restored SOD2 activity. As a whole, this study reports that TGF-β1/miR-382-5p/SOD2 axis deregulation in PMF cells is linked to ROS overproduction that may contribute to enhanced oxidative stress and inflammation. Our results suggest that galunisertib may represent an effective drug reducing abnormal oxidative stress induced by TGF-β1 in PMF patients. Database linking: GEO: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103464.
2017
- Analytic and Dynamic Secretory Profile of Patient-Derived Cytokine-Induced Killer Cells
[Articolo su rivista]
Mesiano, Giulia; Zini, Roberta; Montagner, Giulia; Bianchi, Nicoletta; Manfredini, Rossella; Chillemi, Antonella; Aglietta, Massimo; Grignani, Giovanni; Lampronti, Ilaria; Fiorino, Erika; Malavasi, Fabio; Sangiolo, Dario; Gambari, Roberto; Ferrari, Davide
abstract
Adoptive immunotherapy with cytokine induced killer (CIK) cells has shown antitumor activity against several kinds of cancer in preclinical models and clinical trials. CIK cells are a subset of ex vivo expanded T lymphocytes with T-NK phenotype and MHC-unrestricted antitumor activity. The literature provides scant information on cytokines, chemokines and growth factors secreted by CIK cells. Therefore, we investigated the secretory profile of CIK cells generated from tumor patients. The secretome analysis was performed at specific time points (d 1, d 14 and d 21) of CIK cell expansion. Mature CIK cells (d 21) produce a great variety of interleukins and secreted proteins that can be divided into three groups based on their secretion quantity: high (interleukin [IL]-13, regulated on activation normal T cell expressed and secreted [RANTES] chemokine, MIP-1 alpha and 1 beta), medium (IL-1Ra, IL-5, IL-8, IL-10, IL-17, IP-10, INF-gamma, vascular endothelial growth factor [VEGF] and granulocyte-macrophage colony-stimulating factor [GM-CSF]) and low (IL-1 beta, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, eotaxin, platelet-derived growth factor-bb, basic fibroblast growth factor, G-CSF and monocyte chemoattractant protein [MCP]-1). Moreover, comparing peripheral blood mononuclear cells (PBMCs) (d 1) and mature CIK cells (d 14 and 21) secretomes, we observed that IL-5, IL-10, IL-13, GM-CSF and VEGF were greatly upregulated, while IL-1 beta, IL-6, IL-8, IL-15, IL-17, eotaxin, MCP-1 and RANTES were downregulated. We also performed a gene expression profile analysis of patient-derived CIK cells, showing that mRNA for the different cytokines and secreted proteins was modulated during PBMC-to-CIK differentiation. We highlight previously unknown secretory properties and provide, for the first time, a comprehensive molecular characterization of CIK cells. Our findings provide a rationale to explore the functional implications and possible therapeutic modulation of CIK secretome.
2017
- CALR mutational status identifies different disease subtypes of essential thrombocythemia showing distinct expression profiles
[Articolo su rivista]
Zini, Roberta; Guglielmelli, Paola; Pietra, Daniela; Rumi, Elisa; Rossi, Chiara; Rontauroli, Sebastiano; Genovese, Elena; Fanelli, Tiziana; Calabresi, Laura; Bianchi, Elisa; Salati, Simona; Cazzola, Mario; Tagliafico, Enrico; Vannucchi, Alessandro M.; Manfredini, Rossella
abstract
Polycythemia vera (PV) and essential thrombocythemia (ET) are Philadelphia-negative myeloproliferative neoplasms (MPNs) characterized by erythrocytosis and thrombocytosis, respectively. Approximately 95% of PV and 50-70% of ET patients harbor the V617F mutation in the exon 14 of JAK2 gene, while about 20-30% of ET patients carry CALRins5 or CALRdel52 mutations. These ET CALR-mutated subjects show higher platelet count and lower thrombotic risk compared to JAK2-mutated patients. Here, we showed that CALR-mutated and JAK2V617F-positive CD34+ cells display different gene and miRNA expression profiles. Indeed, we highlighted several pathways differentially activated between JAK2V617F- and CALR-mutated progenitors, i.e., mTOR, MAPK/PI3K, and MYC pathways. Furthermore, we unveiled that the expression of several genes involved in DNA repair, chromatin remodeling, splicing, and chromatid cohesion are decreased in CALR-mutated cells. According to the low risk of thrombosis in CALR-mutated patients, we also found the downregulation of several genes involved in thrombin signaling and platelet activation. As a whole, these data support the model that CALR-mutated ET could be considered as a distinct disease entity from JAK2V617F-positive MPNs and may provide the molecular basis supporting the different clinical features of these patients.
2017
- Deregulated expression of miR-29a-3p, miR-494-3p and miR-660-5p affects sensitivity to tyrosine kinase inhibitors in CML leukemic stem cells
[Articolo su rivista]
Salati, Simona; Salvestrini, Valentina; Carretta, Chiara; Genovese, Elena; Rontauroli, Sebastiano; Zini, Roberta; Rossi, Chiara; Ruberti, Samantha; Bianchi, Elisa; Barbieri, Greta; Curti, Antonio; Castagnetti, Fausto; Gugliotta, Gabriele; Rosti, Gianantonio; Bergamaschi, Micaela; Tafuri, Agostino; Tagliafico, Enrico; Lemoli, Roberto; Manfredini, Rossella
abstract
The development of Imatinib mesylate (IM), which targets the oncogenic BCRABL fusion protein, has greatly improved the outcome of Chronic Myeloid Leukemia (CML) patients. However, BCR-ABL-positive progenitors can be detected in CML patients in complete cytogenetic response. Several evidence suggests that CML stem cells are intrinsically resistant to Tyrosine Kinase Inhibitors (TKI), and therefore they represent the most likely candidate responsible for disease relapse. In this work, we investigated the microRNA (miRNA) expression profile of different subpopulations of CML Leukemic Stem Cells (LSCs): Lin-CD34+CD38-and Lin-CD34-CD38-cells. These cell fractions have been previously shown to be endowed with TKI intrinsic resistance. Our analysis identified 33 common deregulated miRNAs in CML LSCs. Among those, 8 miRNAs were deregulated in CML independently from BCR-ABL kinase activity and therefore are likely to be involved in the BCR-ABL-independent resistance to TKI that characterizes CML LSCs. In particular, the up-regulation of miR-29a-3p and miR-660-5p observed in CML LSCs, led to the down-regulation of their respective targets TET2 and EPAS1 and conferred TKI-resistance to CML LSCs in vitro. On the other hand, miR-494-3p down-regulation in CML LSCs, leading to c-MYC up-regulation, was able to decrease TKI-induced apoptosis. These results demonstrate that aberrant miRNA expression in CML LSCs could contribute to the intrinsic TKIresistance observed in these cell populations, and support the development of novel therapies aimed at targeting aberrantly regulated miRNAs or their targets in order to effectively eradicate CML LSCs.
2017
- Molecular and functional characterization of CD133+ stem/progenitor cells infused in patients with end-stage liver disease reveals their interplay with stromal liver cells
[Articolo su rivista]
Catani, Lucia; Sollazzo, Daria; Bianchi, Elisa; Ciciarello, Marilena; Antoniani, Chiara; Foscoli, Licia; Caraceni, Paolo; Giannone, Ferdinando Antonino; Baldassarre, Maurizio; Giordano, Rosaria; Montemurro, Tiziana; Montelatici, Elisa; D'Errico, Antonia; Andreone, Pietro; Giudice, Valeria; Curti, Antonio; Manfredini, Rossella; Lemoli, Roberto Massimo
abstract
Background aims Growing evidence supports the therapeutic potential of bone marrow (BM)-derived stem/progenitor cells for end-stage liver disease (ESLD). We recently demonstrated that CD133+ stem/progenitor cell (SPC) reinfusion in patients with ESLD is feasible and safe and improve, albeit transiently, liver function. However, the mechanism(s) through which BM-derived SPCs may improve liver function are not fully elucidated. Methods Here, we characterized the circulating SPCs compartment of patients with ESLD undergoing CD133+ cell therapy. Next, we set up an in vitro model mimicking SPCs/liver microenvironment interaction by culturing granulocyte colony-stimulating factor (G-CSF)-mobilized CD133+and LX-2 hepatic stellate cells. Results We found that patients with ESLD show normal basal levels of circulating hematopoietic and endothelial progenitors with impaired clonogenic ability. After G-CSF treatment, patients with ESLD were capable to mobilize significant numbers of functional multipotent SPCs, and interestingly, this was associated with increased levels of selected cytokines potentially facilitating SPC function. Co-culture experiments showed, at the molecular and functional levels, the bi-directional cross-talk between CD133+ SPCs and human hepatic stellate cells LX-2. Human hepatic stellate cells LX-2 showed reduced activation and fibrotic potential. In turn, hepatic stellate cells enhanced the proliferation and survival of CD133+ SPCs as well as their endothelial and hematopoietic function while promoting an anti-inflammatory profile. Discussion We demonstrated that the interaction between CD133+ SPCs from patients with ESLD and hepatic stellate cells induces significant functional changes in both cellular types that may be instrumental for the improvement of liver function in cirrhotic patients undergoing cell therapy.
2017
- Role of miR-34a-5p in Hematopoietic Progenitor Cells Proliferation and Fate Decision: Novel Insights into the Pathogenesis of Primary Myelofibrosis
[Articolo su rivista]
Bianchi, Elisa; Ruberti, Samantha; Rontauroli, Sebastiano; Guglielmelli, Paola; Salati, Simona; Rossi, Chiara; Zini, Roberta; Tagliafico, Enrico; Vannucchi, Alessandro Maria; Manfredini, Rossella
abstract
Primary Myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by a skewed megakaryopoiesis and an overproduction of proinflammatory and profibrotic mediators that lead to the development of bone marrow (BM) fibrosis. Since we recently uncovered the upregulation of miR-34a-5p in PMF CD34+ hematopoietic progenitor cells (HPCs), in order to elucidate its role in PMF pathogenesis here we unravelled the effects of miR-34a-5p overexpression in HPCs. We showed that enforced expression of miR-34a-5p partially constrains proliferation and favours the megakaryocyte and monocyte/macrophage commitment of HPCs. Interestingly, we identified lymphoid enhancer-binding factor 1 (LEF1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts as miR-34a-5p-targets downregulated after miR-34a-5p overexpression in HPCs as well as in PMF CD34+ cells. Remarkably, the knockdown of NR4A2 in HPCs mimicked the antiproliferative effects of miR-34a-5p overexpression, while the silencing of LEF1 phenocopied the effects of miR-34a-5p overexpression on HPCs lineage choice, by favouring the megakaryocyte and monocyte/macrophage commitment. Collectively our data unravel the role of miR-34a-5p in HPCs fate decision and suggest that the increased expression of miR-34a-5p in PMF HPCs could be important for the skewing of megakaryopoiesis and the production of monocytes, that are key players in BM fibrosis in PMF patients.
2017
- miR-494-3p overexpression promotes megakaryocytopoiesis in primary myelofibrosis hematopoietic stem/progenitor cells by targeting SOCS6
[Articolo su rivista]
Rontauroli, Sebastiano; Norfo, Ruggiero; Pennucci, Valentina; Zini, Roberta; Ruberti, Samantha; Bianchi, Elisa; Salati, Simona; Prudente, Zelia; Rossi, Chiara; Rosti, Vittorio; Guglielmelli, Paola; Barosi, Giovanni; Vannucchi, Alessandro; Tagliafico, Enrico; Manfredini, Rossella
abstract
Primary myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by hematopoietic stem cell-derived clonal myeloproliferation, involving especially the megakaryocyte lineage. To better characterize how the altered expression of microRNAs might contribute to PMF pathogenesis, we have previously performed the integrative analysis of gene and microRNA expression profiles of PMF hematopoietic stem/progenitor cells (HSPCs), which allowed us to identify miR- 494-3p as the upregulated microRNA predicted to target the highest number of downregulated mRNAs. To elucidate the role of miR-494-3p in hematopoietic differentiation, in the present study we demonstrated that miR-494-3p enforced expression in normal HSPCs promotes megakaryocytopoiesis. Gene expression profiling upon miR-494-3p overexpression allowed the identification of genes commonly downregulated both after microRNA overexpression and in PMF CD34+ cells. Among them, suppressor of cytokine signaling 6 (SOCS6) was confirmed to be a miR-494-3p target by luciferase assay. Western blot analysis showed reduced level of SOCS6 protein as well as STAT3 activation in miR-494-3p overexpressing cells. Furthermore, transient inhibition of SOCS6 expression in HSPCs demonstrated that SOCS6 silencing stimulates megakaryocytopoiesis, mimicking the phenotypic effects observed upon miR-494-3p overexpression. Finally, to disclose the contribution of miR-494-3p upregulation to PMF pathogenesis, we performed inhibition experiments in PMF HSPCs, which showed that miR-494-3p silencing led to SOCS6 upregulation and impaired megakaryocyte differentiation. Taken together, our results describe for the first time the role of miR-494- 3p during normal HSPC differentiation and suggest that its increased expression, and the subsequent downregulation of its target SOCS6, might contribute to the megakaryocyte hyperplasia commonly observed in PMF patients.
2016
- A data-driven network model of primary myelofibrosis: transcriptional and post-transcriptional alterations in CD34+ cells
[Articolo su rivista]
Calura, E.; Pizzini, S.; Bisognin, A.; Coppe, A.; Sales, G.; Gaffo, E.; Fanelli, T.; Mannarelli, C.; Zini, Roberta; Norfo, Ruggiero; Pennucci, Valentina; Manfredini, Rossella; Romualdi, C.; Guglielmelli, P.; Vannucchi, A. M.; Bortoluzzi, Stefania; Associazione italiana per la ricerca sul cancro Gruppo Italiano Malattie Mieloproliferative, Investigators
abstract
microRNAs (miRNAs) are relevant in the pathogenesis of primary myelofibrosis (PMF) but our understanding is limited to specific target genes and the overall systemic scenario islacking. By both knowledge-based and ab initio approaches for comparative analysis of CD34+ cells of PMF patients and healthy controls, we identified the deregulated pathways involving miRNAs and genes and new transcriptional and post-transcriptional regulatory circuits in PMF cells. These converge in a unique and integrated cellular process, in which the role of specific miRNAs is to wire, co-regulate and allow a fine crosstalk between the involved processes. The PMF pathway includes Akt signaling, linked to Rho GTPases, CDC42, PLD2, PTEN crosstalk with the hypoxia response and Calcium-linked cellular processes connected to cyclic AMP signaling. Nested on the depicted transcriptional scenario, predicted circuits are reported, opening new hypotheses. Links between miRNAs (miR-106a-5p, miR-20b-5p, miR-20a-5p, miR-17-5p, miR-19b-3p and let-7d-5p) and key transcription factors (MYCN, ATF, CEBPA, REL, IRF and FOXJ2) and their common target genes tantalizingly suggest new path to approach the disease. The study provides a global overview of transcriptional and post-transcriptional deregulations in PMF, and, unifying consolidated and predicted data, could be helpful to identify new combinatorial therapeutic strategy. Interactive PMF network model: http://compgen.bio.unipd.it/pmf-net/.
2016
- Epidemiology and clinical relevance of mutations in postpolycythemia vera and postessential thrombocythemia myelofibrosis: A study on 359 patients of the AGIMM group
[Articolo su rivista]
Rotunno, Giada; Pacilli, Annalisa; Artusi, Valentina; Rumi, Elisa; Maffioli, Margherita; Delaini, Federica; Brogi, Giada; Fanelli, Tiziana; Pancrazzi, Alessandro; Pietra, Daniela; Bernardis, Isabella; Belotti, Clara; Pieri, Lisa; Sant'Antonio, Emanuela; Salmoiraghi, Silvia; Cilloni, Daniela; Rambaldi, Alessandro; Passamonti, Francesco; Barbui, Tiziano; Manfredini, Rossella; Cazzola, Mario; Tagliafico, Enrico; Vannucchi, Alessandro M; Guglielmelli, Paola
abstract
Transformation to secondary myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PPV-MF) and essential thrombocythemia (PET-MF). Although primary (PMF) and secondary MF are considered similar diseases and managed similarly, there are few studies specifically focused on the latter. The aim of this study was to characterize the mutation landscape, and describe the main clinical correlates and prognostic implications of mutations, in a series of 359 patients with PPV-MF and PET-MF. Compared with PV and ET, the JAK2V617F and CALR mutated allele burden was significantly higher in PPV-MF and/or PET-MF, indicating a role for accumulation of mutated alleles in the process of transformation to MF. However, neither the allele burden nor the type of driver mutation influenced overall survival (OS), while absence of any driver mutation (triple negativity) was associated with significant reduction of OS in PET-MF, similar to PMF. Of the five interrogated subclonal mutations (ASXL1, EZH2, SRSF2, IDH1, and IDH2), that comprise a prognostically detrimental high molecular risk (HMR) category in PMF, only SRSF2 mutations were associated with reduced survival in PET-MF, and no additional mutation profile with prognostic relevance was highlighted. Overall, these data indicate that the molecular landscape of secondary forms of MF is different from PMF, suggesting that unknown mutational events might contribute to the progression from chronic phase disease to myelofibrosis. These findings also support more extended genotyping approaches aimed at identifying novel molecular abnormalities with prognostic relevance for patients with PPV-MF and PET-MF. Am. J. Hematol. 91:681–686, 2016. © 2016 Wiley Periodicals, Inc.
2016
- Genomic landscape of megakaryopoiesis and platelet function defects
[Articolo su rivista]
Bianchi, Elisa; Norfo, Ruggiero; Pennucci, Valentina; Zini, Roberta; Manfredini, Rossella
abstract
Megakaryopoiesis is a complex, stepwise process that takes place largely in the bone marrow. At the apex of the hierarchy, hematopoietic stem cells undergo a number of lineage commitment decisions that ultimately lead to the production of polyploid megakaryocytes. On average, megakaryocytes release 1011 platelets per day into the blood that repair vascular injuries and prevent excessive bleeding. This differentiation process is tightly controlled by exogenous and endogenous factors, which have been the topics of intense research in the hematopoietic field. Indeed, a skewing of megakaryocyte commitment and differentiation may entail the onset of myeloproliferative neoplasms and other preleukemic disorders together with acute megakaryoblastic leukemia, whereas quantitative or qualitative defects in platelet production can lead to inherited platelet disorders. The recent advent of next-generation sequencing has prompted mapping of the genomic landscape of these conditions to provide an accurateview of the underlying lesions. The aims of this review are to introduce the physiological pathways of megakaryopoiesis and to present landmark studies on acquired and inherited disorders that target them. These studies have not only introduced a new era in the fields of molecular medicine and targeted therapies but may also provide us with a better understanding ofthemechanismsunderlying normalmegakaryopoiesis and thrombopoiesis that can informeffortsto create alternativesources of megakaryocytes and platelets.
2016
- Integrative analysis of copy number and gene expression data suggests novel pathogenetic mechanisms in primary myelofibrosis
[Articolo su rivista]
Salati, Simona; Zini, Roberta; Nuzzo, Simona; Guglielmelli, Paola; Pennucci, Valentina; Prudente, Zelia; Ruberti, Samantha; Rontauroli, Sebastiano; Norfo, Ruggiero; Bianchi, Elisa; Bogani, Costanza; Rotunno, Giada; Fanelli, Tiziana; Mannarelli, Carmela; Rosti, Vittorio; Salmoiraghi, Silvia; Pietra, Daniela; Ferrari, Sergio; Barosi, Giovanni; Rambaldi, Alessandro; Cazzola, Mario; Bicciato, Silvio; Tagliafico, Enrico; Vannucchi, Alessandro M; Manfredini, Rossella
abstract
Primary myelofibrosis (PMF) is a Myeloproliferative Neoplasm (MPN) characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and transformation to Acute Myeloid Leukemia (AML). A number of phenotypic driver (JAK2, CALR, MPL) and additional subclonal mutations have been described in PMF, pointing to a complex genomic landscape. To discover novel genomic lesions that can contribute to disease phenotype and/or development, gene expression and copy number signals were integrated and several genomic abnormalities leading to a concordant alteration in gene expression levels were identified. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus was accompanied by a coordinated transcriptional up-regulation in PMF patients. PAOX inhibition resulted in rapid cell death of PMF progenitor cells, while sparing normal cells, suggesting that PAOX inhibition could represent a therapeutic strategy to selectively target PMF cells without affecting normal hematopoietic cells' survival. Moreover, copy number loss in the chromatin modifier HMGXB4 gene correlates with a concomitant transcriptional down-regulation in PMF patients. Interestingly, silencing of HMGXB4 induces megakaryocyte differentiation, while inhibiting erythroid development, in human hematopoietic stem/progenitor cells. These results highlight a previously un-reported, yet potentially interesting role of HMGXB4 in the hematopoietic system and suggest that genomic and transcriptional imbalances of HMGXB4 could contribute to the aberrant expansion of the megakaryocytic lineage that characterizes PMF patients.
2016
- The isopeptidase inhibitor 2cPE triggers proteotoxic stress and ATM activation in chronic lymphocytic leukemia cells
[Articolo su rivista]
Tomasella, Andrea; Picco, Raffaella; Ciotti, Sonia; Sgorbissa, Andrea; Bianchi, Elisa; Manfredini, Rossella; Benedetti, Fabio; Trimarco, Valentina; Frezzato, Federica; Trentin, Livio; Semenzato, Gianpietro; Delia, Domenico; Brancolini, Claudio
abstract
Relapse after treatment is a common and unresolved problem for patients suffering of the B-cell chronic lymphocytic leukemia (B-CLL). Here we investigated the ability of the isopeptidase inhibitor 2cPE to trigger apoptosis in leukemia cells in comparison with bortezomib, another inhibitor of the ubiquitin-proteasome system (UPS). Both inhibitors trigger apoptosis in CLL B cells and gene expression profiles studies denoted how a substantial part of genes up-regulated by these compounds are elements of adaptive responses, aimed to sustain cell survival. 2cPE treatment elicits the up-regulation of chaperones, proteasomal subunits and elements of the anti-oxidant response. Selective inhibition of these responses augments apoptosis in response to 2cPE treatment. We have also observed that the product of the ataxia telangiectasia mutated gene (ATM) is activated in 2cPE treated cells. Stimulation of ATM signaling is possibly dependent on the alteration of the redox homeostasis. Importantly ATM inhibition, mutations or down-modulation increase cell death in response to 2cPE. Overall this work suggests that 2cPE could offer new opportunities for the treatment of B-CLL.
2016
- Tie2 expressing monocytes in the spleen of patients with primary myelofibrosis
[Articolo su rivista]
Campanelli, R.; Fois, G.; Catarsi, P.; Poletto, V.; Villani, L.; Erba, B. G.; Maddaluno, L.; Jemos, B.; Salmoiraghi, S.; Guglielmelli, P.; Abbonante, V.; Di Buduo, C. A.; Balduini, A.; Iurlo, A.; Barosi, G.; Rosti, V.; Massa, M.; Vannucchi, A. M.; Balliu, M.; Bartalucci, N.; Bogani, C.; Bosi, A.; Calabresi, L.; Corbizzi Fattori, G.; Fanelli, T.; Fjerza, R.; Gesullo, F.; Mannarelli, C.; Merli, L.; Pacilli, A.; Pancrazzi, A.; Paoli, C.; Pieri, L.; Rotunno, G.; Sant'Antonio, E.; Bonetti, E.; Cazzola, M.; Ambaglio, I.; Bernasconi, P.; Casetti, C. I.; Catricala, S.; Elena, C.; Fugazza, E.; Galli, A.; Malcovati, L.; Milanesi, C.; Pascutto, C.; Pietra, D.; Ripamonti, F.; Rossi, M.; Rumi, E.; Dejana, E.; Breviario, F.; Corada, M.; Malinverno, M.; Rambaldi, A.; Chioda, G.; Ferrari, M. L.; Finazzi, G.; Finazzi, M. C.; Belotti, C.; Boroni, C.; Amaru, A.; Golay, J.; Bortoluzzi, S.; Bisognin, A.; Coppe, A.; Saccoman, C.; Manfredini, R.; Artuso, L.; Bernardis, I.; Bianchi, E.; Montanari, M.; Pennucci, V.; Prudente, Z.; Rontauroli, S.; Rossi, C.; Ruberti, S.; Salati, S.; Tagliafico, E.; Tenedini, E.; Zini, R.
abstract
Primary myelofibrosis (PMF) is a Philadelphia-negative (Ph-) myeloproliferative disorder, showing abnormal CD34 + progenitor cell trafficking, splenomegaly, marrow fibrosis leading to extensive extramedullary haematopoiesis, and abnormal neoangiogenesis in either the bone marrow or the spleen. Monocytes expressing the angiopoietin-2 receptor (Tie2) have been shown to support abnormal angiogenic processes in solid tumors through a paracrine action that takes place in proximity to the vessels. In this study we investigated the frequency of Tie2 expressing monocytes in the spleen tissue samples of patients with PMF, and healthy subjects (CTRLs), and evaluated their possible role in favouring spleen angiogenesis. We show by confocal microscopy that in the spleen tissue of patients with PMF, but not of CTRLs, the most of the CD14 + cells are Tie2 + and are close to vessels; by flow cytometry, we found that Tie2 expressing monocytes were Tie2 + CD14 low CD16 bright CDL62 - CCR2 - (TEMs) and their frequency was higher (p = 0.008) in spleen tissue-derived mononuclear cells (MNCs) of patients with PMF than in spleen tissue-derived MNCs from CTRLs undergoing splenectomy for abdominal trauma. By in vitro angiogenesis assay we evidenced that conditioned medium of immunomagnetically selected spleen tissue derived CD14 + cells of patients with PMF induced a denser tube like net than that of CTRLs; in addition, CD14 + Tie2 + cells sorted from spleen tissue derived single cell suspension of patients with PMF show a higher expression of genes involved in angiogenesis than that found in CTRLs. Our results document the enrichment of Tie2 + monocytes expressing angiogenic genes in the spleen of patients with PMF, suggesting a role for these cells in starting/maintaining the pathological angiogenesis in this organ.
2016
- Unravelling the Complexity of Inherited Retinal Dystrophies Molecular Testing: Added Value of Targeted Next-Generation Sequencing
[Articolo su rivista]
Bernardis, Isabella; Chiesi, Laura; Tenedini, Elena; Artuso, Lucia; Percesepe, Antonio; Artusi, Valentina; Simone, Maria Luisa; Manfredini, Rossella; Camparini, Monica; Rinaldi, Chiara; Ciardella, Antonio; Graziano, Claudio; Balducci, Nicole; Tranchina, Antonia; Cavallini, Gian Maria; Pietrangelo, Antonello; Marigo, Valeria; Tagliafico, Enrico
abstract
To assess the clinical utility of targeted Next-Generation Sequencing (NGS) for the diagnosis of Inherited Retinal Dystrophies (IRDs), a total of 109 subjects were enrolled in the study, including 88 IRD affected probands and 21 healthy relatives. Clinical diagnoses included Retinitis Pigmentosa (RP), Leber Congenital Amaurosis (LCA), Stargardt Disease (STGD), Best Macular Dystrophy (BMD), Usher Syndrome (USH), and other IRDs with undefined clinical diagnosis. Participants underwent a complete ophthalmologic examination followed by genetic counseling. A custom AmpliSeq� panel of 72 IRD-related genes was designed for the analysis and tested using Ion semiconductor Next-Generation Sequencing (NGS). Potential disease-causing mutations were identified in 59.1% of probands, comprising mutations in 16 genes. The highest diagnostic yields were achieved for BMD, LCA, USH, and STGD patients, whereas RP confirmed its high genetic heterogeneity. Causative mutations were identified in 17.6% of probands with undefined diagnosis. Revision of the initial diagnosis was performed for 9.6% of genetically diagnosed patients. This study demonstrates that NGS represents a comprehensive cost-effective approach for IRDs molecular diagnosis. The identification of the genetic alterations underlying the phenotype enabled the clinicians to achieve a more accurate diagnosis. The results emphasize the importance of molecular diagnosis coupled with clinic information to unravel the extensive phenotypic heterogeneity of these diseases.
2016
- miR-382-5p Controls Hematopoietic Stem Cell Differentiation Through the Downregulation of MXD1
[Articolo su rivista]
Zini, Roberta; Rossi, Chiara; Norfo, Ruggiero; Pennucci, Valentina; Barbieri, Greta; Ruberti, Samantha; Rontauroli, Sebastiano; Salati, Simona; Bianchi, Elisa; Manfredini, Rossella
abstract
microRNAs are key regulators of gene expression that control stem cell fate by posttranscriptional downregulation of hundreds of target genes through seed pairing in their 3' untranslated region. In fact, miRNAs tightly regulate fundamental stem cell processes, like self-renewal, proliferation, and differentiation; therefore, miRNA deregulation may contribute to the development of solid tumors and hematological malignancies. miR-382-5p has been found to be upregulated in patients with myeloid neoplasms, but its role in normal hematopoiesis is still unknown. In this study, we demonstrated that miR-382-5p overexpression in CD34(+) hematopoietic stem/progenitor cells (HSPCs) leads to a significant decrease of megakaryocyte precursors coupled to increase of granulocyte ones. Furthermore, by means of a computational analysis using different prediction algorithms, we identified several putative mRNA targets of miR-382-5p that are downregulated upon miRNA overexpression (ie, FLI1, GATA2, MAF, MXD1, RUNX1, and SGK1). Among these, we validated MXD1 as real target of miR-382-5p by luciferase reporter assay. Finally, we showed that MXD1 knockdown mimics the effects of miR-382-5p overexpression on granulocyte and megakaryocyte differentiation of CD34(+) cells. Overall, our results demonstrated that miR-382-5p expression favors the expansion of granulocyte lineage and impairs megakaryocyte commitment through MXD1 downregulation. Therefore, our data showed for the first time that the miR-382-5p/MXD1 axis plays a critical role in myelopoiesis by affecting the lineage choice of CD34(+) HSPCs.
2015
- AMPLICON-BASED NGS: AN EFFECTIVE APPROACH FOR THE MOLECULAR DIAGNOSIS OF EPIDERMOLYSIS BULLOSA
[Abstract in Atti di Convegno]
Tenedini, Elena; Artuso, Lucia; Bernardis, Isabella; Artusi, Valentina; Percesepe, A; DE ROSA, Laura; Contin, Roberta; Manfredini, Rossella; Pellacani, Giovanni; Giannetti, A; DE LUCA, Michele; Tagliafico, Enrico
abstract
Background: Epidermolysis Bullosa (EB) is caused by mutations in genes that encode proteins belonging to the epidermal-dermal
junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, the current methods available
for diagnosing EB involve immunohistochemistry of bioptic samples and transmission electron microscopy followed
by single candidate gene Sanger Sequencing (SS), which are labour intensive and expensive clinical pathways.
Objectives: According to the recently published recommendations for the EB diagnosis and treatment, the assessment of
the mutational landscape is now a fundamental step for developing a comprehensive diagnostic path. Next-Generation
Sequencing (NGS) via the parallel ultra-deep sequencing of many genes represents a proper method for reducing the
processing time and costs of EB diagnostics.
Methods: We developed an EB disease-comprehensive AmpliSeq panel to accomplish the NGS on the Ion Torrent PGM
platform. The panel was performed on ten patients with known genetic diagnoses and was then employed in eight family
trios with unknown molecular footprints.
Results: The panel was successful in finding the causative mutations in all ten of the patients with known mutations, fully
confirming the SS data and providing proof of concept of the sensitivity, specificity, and accuracy of this procedure. In
addition to being consistent with the clinical diagnosis, it was also effective in the trios, identifying all of the variants, including
ones that the SS missed or de novo mutations.
Conclusions: The NGS and AmpliSeq were shown to be an effective approach for the diagnosis of EB, resulting in a costand
time-effective 72-hour procedure.
2015
- Abnormal expression patterns of WT1-as, MEG3 and ANRIL long non-coding RNAs in CD34+ cells from patients with primary myelofibrosis and their clinical correlations
[Articolo su rivista]
Pennucci, Valentina; Zini, Roberta; Norfo, Ruggiero; Guglielmelli, Paola; Bianchi, Elisa; Salati, Simona; Sacchi, Giorgia; Prudente, Zelia; Tenedini, Elena; Ruberti, Samantha; Paoli, Chiara; Fanelli, Tiziana; Mannarelli, Carmela; Tagliafico, Enrico; Ferrari, Sergio; Vannucchi, ALESSANDRO MARIA; Manfredini, Rossella; Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative, Investigators
abstract
Abnormal expression patterns of WT1-as, MEG3 and ANRIL long non-coding RNAs in CD34+ cells from patients with primary myelofibrosis and their clinical correlations.
2015
- Amplicon-based Next Generation Sequencing: an effective approach to molecular diagnosis of Epidermolysis Bullosa
[Abstract in Atti di Convegno]
Tenedini, Elena; Artuso, Lucia; Bernardis, Isabella; Artusi, Valentina; Percesepe, Antonio; Manfredini, Rossella; DE ROSA, Laura; Contin, Roberta; Pellacani, Giovanni; Giannetti, Alberto; Pagani, Jacopo; DE LUCA, Michele; Tagliafico, Enrico
abstract
Epidermolysis Bullosa (EB) is caused by mutations in genes encoding for proteins of the epidermal–dermal junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, current methods in EB diagno- stics comprise immunohistochemistry on bioptic samples and transmission electron microscopy followed by single candidate gene Sanger Sequencing (SS) that therefore represents the final phase of a labour intensive and ex- pensive clinical pathway.
Methods: Participants in a cross sectional study included individuals with Muenke syndrome (P250R mutation in FGFR3) and their mutation negative siblings. Participants completed validated assessments of executive functio- ning (Behavior Rating Inventory of Executive Function; BRIEF) and adaptive behavior skills (Adaptive Behavior Assessment System; ABAS-II).
According to the recently published recommendations for diagnosis and treatment in EB, the assessment of mutational landscape is instead a fun- damental step to a comprehensive diagnosis path; Next Generation Sequen- cing (NGS), throughout parallel ultra-deep sequencing of many genes, would represent a proper method for reducing timing and costs in EB diagnostics. We developed an EB disease-comprehensive amplicon panel (AmpliSeq pa- nel), to accomplish NGS onto Ion Torrent PGM platform. The panel was dealt on ten patients with known genetic diagnosis, and then employed in eight family trios with unknown molecular footprinting.
Results: Forty-four FGFR3 mutation positive individuals, median age 9, range 6 months to 52 years were evaluated with the BRIEF and ABAS-II. Additio- nally, 10 unaffected siblings were used as controls. For the General Executive Composite scale of the BRIEF, 32.1% of the cohort had scores greater than +1.5 SD, signifying “Potential Clinical Significance.” For the General Adaptive Composite of the ABAS-II, 28.2% of affected individuals scored in the “Ex- tremely Low” category” (3rd -8th percentile of normative population) and 53.9% were below the “Average” category (less than the 25th percentile). Multiple regression analysis showed that the presence of craniosynostosis was not a predictor (P = 0.7) of BRIEF and ABAS-II scores.
The AmpliSeq panel, obtaining a proof of concept of the sensitivity, specificity, and accuracy of this kind of procedure, showed successful in finding the causative mutations in all the ten patients with known mutations, fully confirming SS data. Besides, showing consistent with the clinical diagnosis, it was effective in trios, identifying all the variants, even the ones SS missed or in case of de novo mutations. NGS
2015
- Amplicon-based next-generation sequencing: an effective approach for the molecular diagnosis of epidermolysis bullosa
[Articolo su rivista]
Tenedini, Elena; Artuso, Lucia; Bernardis, Isabella; Artusi, Valentina; Percesepe, Antonio; De Rosa, Laura; Contin, Roberta; Manfredini, Rossella; Pellacani, Giovanni; Giannetti, Alberto; Pagani, J.; De Luca, Michele; Tagliafico, Enrico
abstract
Epidermolysis bullosa (EB) is caused by mutations in genes that encode proteins belonging to the epidermal-dermal junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, the current methods available for diagnosing EB involve immunohistochemistry of biopsy samples and transmission electron microscopy followed by single-candidate gene Sanger sequencing (SS), which are labour-intensive and expensive clinical pathways.
2015
- Implementation of an NGS-based workflow for BRCA1 and BRCA2 mutation screening
[Abstract in Atti di Convegno]
Artuso, Lucia; Medici, Veronica; Bernardis, Isabella; Tenedini, Elena; Artusi, Valentina; Simone, Maria Luisa; Tarugi, Patrizia Maria; Manfredini, Rossella; Cortesi, Laura; Tagliafico, Enrico
abstract
probability to develop familiar breast cancer. To detect BRCA1/2 germline mutations we developed a next-generation sequencing (NGS) routine dia- gnostic workflow, based on the Ion Torrent PGMTM System platform. The Ion AmpliSeqTM BRCA1 and BRCA2 Community Panel was handled with a semi- automatized procedure for multiplex PCR-based library preparation and se- quencing. Data analysis required the implementation of a custom designed bioinformatic pipeline for sequences alignment and for the identification, annotation and filtration of genetic variants. Sanger sequencing was perfor- med to validate candidate mutations, and to re-sequence amplicons having low NGS coverage (<50 reads per amplicon). Negative samples were ana- lyzed using the BRCA HP Kit (Multiplicom) for an effective homopolymeric stretches detection. This workflow together with the potentiality of our bio- informatic pipeline was blindly tested and validated onto a small cohort of patients previously Sanger sequenced, fine-tuning the parameter settings and resulting in a sensitivity of 100% in variant detection. Subsequently, 244 patients were analyzed thus confirming the need of a double check for the homopolymeric stretches with both NGS sequencing and BRCA HP Kit. The NGS-based workflow here proposed was able to decrease the overall
2015
- MYB controls erythroid versus megakaryocyte lineage fate decision through the miR-486-3p-mediated downregulation of MAF
[Articolo su rivista]
Bianchi, E.; Bulgarelli, J.; Ruberti, S.; Rontauroli, S.; Sacchi, G.; Norfo, R.; Pennucci, V.; Zini, R.; Salati, S.; Prudente, Z.; Ferrari, S.; Manfredini, R.
abstract
The transcription factor MYB has a key role in hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that MYB controls erythroid versus megakaryocyte lineage decision by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which MYB affects lineage fate decision, we performed the integrative analysis of miRNA and mRNA changes in MYB-silenced human primary CD34+ HPCs. Among the miRNAs with the highest number of predicted targets, we focused our studies on hsa-miR-486-3p by demonstrating that MYB controls miR-486-3p expression through the transactivation of its host gene, ankyrin-1 (ANK1) and that miR-486-3p affects HPCs commitment. Indeed, overexpression and knockdown experiments demonstrated that miR-486-3p supports the erythropoiesis while restraining the megakaryopoiesis. Of note, miR-486-3p also favors granulocyte differentiation while repressing the macrophage differentiation. To shed some light on the molecular mechanisms through which miR-486-3p affects HPCs lineage commitment, we profiled the gene expression changes upon miR-486-3p overexpression in CD34+ cells. Among the genes downregulated in miR-486-3p-overexpressing HPCs and computationally predicted to be miR-486-3p targets, we identified MAF as a miR-486-3p target by 3′UTR luciferase reporter assay. Noteworthy, MAF overexpression was able to partially reverse the effects of miR-486-3p overexpression on erythroid versus megakaryocyte lineage choice. Moreover, the MYB/MAF co-silencing constrained the skewing of erythroid versus megakaryocyte lineage commitment in MYB-silenced CD34+ cells, by restraining the expansion of megakaryocyte lineage while partially rescuing the impairment of erythropoiesis. Therefore, our data collectively demonstrate that MYB favors erythropoiesis and restrains megakaryopoiesis through the transactivation of miR-486-3p expression and the subsequent downregulation of MAF. As a whole, our study uncovers the MYB/miR-486-3p/MAF axis as a new mechanism underlying the MYB-driven control of erythroid versus megakaryocyte lineage fate decision.
2014
- C-Myb Restrains Megakaryopoiesis through the Hsa-MiR-486-3p-Driven Down-Regulation of C-Maf
[Abstract in Rivista]
Bianchi, Elisa; Bulgarelli, Jenny; Sacchi, Giorgia; Ruberti, Samantha; Norfo, Ruggiero; Rontauroli, Sebastiano; Pennucci, Valentina; Zini, Roberta; Salati, Simona; Prudente, Zelia; Ferrari, Sergio; Vannucchi, Alessandro M.; Manfredini, Rossella
abstract
INTRODUCTION
The transcription factor c-Myb plays a key role in human primary CD34+ hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that c-Myb affects erythroid versus megakaryocyte lineage decision in part by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which c-myb affects lineage fate decision, we profiled the miRNA and mRNA changes in myb-silenced CD34+ HPCs.
METHODS
RNA from CD34+ HPCs transfected with c-myb-targeting/non targeting control synthetic siRNAs was collected 24 hours post-Nucleofection for a set of 5 independent experiments.
mRNA and miRNA expression for each sample were profiled by Affymetrix U219 GeneAtlas and Exiqon Human miRNome PCR Panel, respectively. miRNA/mRNA data were integrated by Ingenuity Pathway Analysis.
The effects of hsa-miR-486-3p overexpression and c-Maf silencing on CD34+ cells differentiation ability were studied by morphological and immunophenotypic analyses after liquid culture and by collagen-based clonogenic assay. Furthermore, gene expression changes in CD34+ cells upon hsa-miR-486-3p overexpression were profiled by Affymetrix U219.
RESULTS
The integrative analysis of miRNA/mRNA expression changes upon c-myb silencing in human CD34+ HPCs highlighted a set of 19 miRNA with 150 anticorrelated putative target mRNAs. Among the miRNAs down-regulated in myb-silenced progenitors with the highest number of predicted target mRNAs, we selected hsa-miR-486-3p based on the in vitro effects of its overexpression on HPCs commitment. Indeed, morphological and flow cytometric analyses after liquid culture showed that hsa-miR-486-3p overexpression in HPCs enhanced erythroid and granulocyte differentiation while restraining megakaryocyte and macrophage differentiation. Moreover, collagen-based clonogenic assay demonstrated a strong impairement megakaryocyte commitment upon hsa-miR-486-3p-overexpression in CD34+ cells.
Gene expression profiling of hsa-miR-486-3p overexpressing CD34+ cells enabled us to identify a set of 8 genes down-regulated and computationally predicted, putative hsa-miR-486-3p targets. Among them, we selected c-maf transcript as up-regulated upon myb silencing. Worth of note, c-maf silencing in CD34+ progenitor cells was able to reverse the affects of myb silencing on erythroid versus megakaryocyte lineage choice.
CONCLUSIONS
Integrative miRNA/mRNA analysis highlighted a set of miRNAs and anticorrelated putative target mRNAs modulated upon myb silencing, therefore potential players in myb-driven HPCs lineage choice. Among them, we demonstrated the hsa-miR-486-3p/c-maf pair as partially contributing to the effects of myb on HPCs commitment. Therefore, our data collectively identified myb-driven hsa-miR-486-3p up-regulation and subsequent c-maf down-regulation as a new molecular mechanism through which c-Myb favours erythropoiesis while restraining megakaryopoiesis.
2014
- Calreticulin mutation-specific immunostaining in myeloproliferative neoplasms: Pathogenetic insight and diagnostic value
[Articolo su rivista]
Vannucchi, A. M.; Rotunno, G.; Bartalucci, N.; Raugei, G.; Carrai, V.; Balliu, M.; Mannarelli, C.; Pacilli, A.; Calabresi, L.; Fjerza, R.; Pieri, L.; Bosi, A.; Manfredini, Rossella; Guglielmelli, P.
abstract
Mutations in the gene calreticulin (CALR) occur in the majority of JAK2- and MPL-unmutated patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF); identifying CALR mutations contributes to the diagnostic pathway of ET and PMF. CALR mutations are heterogeneous spanning over the exon 9, but all result in a novel common protein C terminus. We developed a polyclonal antibody against a 17-amino-acid peptide derived from mutated calreticulin that was used for immunostaining of bone marrow biopsies. We show that this antibody specifically recognized patients harboring different types of CALR mutation with no staining in healthy controls and JAK2- or MPL-mutated ET and PMF. The labeling was mostly localized in megakaryocytes, whereas myeloid and erythroid cells showed faint staining, suggesting a preferential expression of calreticulin in megakaryocytes. Megakaryocytic-restricted expression of calreticulin was also demonstrated using an antibody against wild-type calreticulin and by measuring the levels of calreticulin RNA by gene expression analysis. Immunostaining using an antibody specific for mutated calreticulin may become a rapid, simple and cost-effective method for identifying CALR-mutated patients complementing molecular analysis; furthermore, the labeling pattern supports the preferential expansion of megakaryocytic cell lineage as a result of CALR mutation in an immature hematopoietic stem cell.
2014
- Impact of mutational status on outcomes in myelofibrosis patients treated with ruxolitinib in the COMFORT-II study
[Articolo su rivista]
Guglielmelli, Paola; Biamonte, Flavia; Rotunno, Giada; Artusi, Valentina; Artuso, Lucia; Bernardis, Isabella; Tenedini, Elena; Pieri, Lisa; Paoli, Chiara; Mannarelli, Carmela; Fjerza, Rajmonda; Rumi, Elisa; Stalbovskaya, Viktoriya; Squires, Matthew; Cazzola, Mario; Manfredini, Rossella; Harrison, Claire; Tagliafico, Enrico; Vannucchi, ALESSANDRO MARIA
abstract
The JAK1/JAK2 inhibitor ruxolitinib produced significant reductions in splenomegaly and symptomatic burden and improved survival in patients with myelofibrosis (MF), irrespective of their JAK2 mutation status, in 2 phase III studies against placebo (COMFORT-I) and best available therapy (COMFORT-II). We performed a comprehensive mutation analysis to evaluate the impact of 14 MF-associated mutations on clinical outcomes in 166 patients included in COMFORT-II. We found that responses in splenomegaly and symptoms, as well as the risk of developing ruxolitinib-associated anemia and thrombocytopenia, occurred at similar frequencies across different mutation profiles. Ruxolitinib improved survival independent of mutation profile and reduced the risk of death in patients harboring a set of prognostically detrimental mutations (ASXL1, EZH2, SRSF2, IDH1/2) with an hazard ratio of 0.57 (95% confidence interval: 0.30-1.08) vs best available therapy. These data indicate that clinical efficacy and survival improvement may occur across different molecular subsets of patients with MF treated with ruxolitinib.
2014
- Targeted cancer exome sequencing reveals recurrent mutations in myeloproliferative neoplasms
[Articolo su rivista]
Tenedini, Elena; Bernardis, Isabella; Artusi, Valentina; Artuso, Lucia; Roncaglia, E.; Guglielmelli, P.; Pieri, L.; Bogani, C.; Biamonte, F.; Rotunno, G.; Mannarelli, C.; Bianchi, Elisa; Pancrazzi, A.; Fanelli, T.; MALAGOLI TAGLIAZUCCHI, Guidantonio; Ferrari, Sergio; Manfredini, Rossella; Vannucchi, A. M.; Tagliafico, Enrico
abstract
With the intent of dissecting the molecular complexity of Philadelphia-negative myeloproliferative neoplasms (MPN), we designed a target enrichment panel to explore, using next-generation sequencing (NGS), the mutational status of an extensive list of 2,000 cancer-associated genes and microRNAs. The genomic DNA of granulocytes and in-vitro-expanded CD3+ T-lymphocytes, as a germline control, was target-enriched and sequenced in a learning cohort of 20 MPN patients using Roche 454 technology. We identified 141 genuine somatic mutations, most of which were not previously described. To test the frequency of the identified variants, a larger validation cohort of 189 MPN patients was additionally screened for these mutations using Ion Torrent AmpliSeq NGS. Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG, and NRAS), we demonstrated a mutation frequency between 3 and 8%.
We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest DIPSS-plus score categories. This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing an NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score.
2014
- The number of prognostically detrimental mutations and prognosis in primary myelofibrosis: An international study of 797 patients
[Articolo su rivista]
Guglielmelli, P; Lasho, T. L.; Rotunno, G.; Score, J.; Mannarelli, C.; Pancrazzi, A.; Biamonte, F.; Pardanani, A.; Zoi, K.; Reiter, A.; Duncombe, A.; Fanelli, T.; Pietra, D.; Rumi, E.; Finke, C.; Gangat, N.; Ketterling, R. P.; Knudson, R. A.; Hanson, C. A.; Bosi, A.; Pereira, A.; Manfredini, Rossella; Cervantes, F.; Barosi, G.; Cazzola, M.; Cross, N. C. P.; Vannucchi, A. M.; Tefferi, A.
abstract
We recently defined a high-molecular risk category (HMR) in primary myelofibrosis (PMF), based on the presence of at least one of the five 'prognostically detrimental' mutated genes (ASXL1, EZH2, SRSF2 and IDH1/2). Herein, we evaluate the additional prognostic value of the 'number' of mutated genes. A total of 797 patients were recruited from Europe (n=537) and the Mayo Clinic (n=260). In the European cohort, 167 (31%) patients were HMR: 127 (23.6%) had one and 40 (7.4%) had two or more mutated genes. The presence of two or more mutations predicted the worst survival: median 2.6 years (hazard ratio (HR) 3.8, 95% confidence interval (CI) 2.6-5.7) vs. 7.0 years (HR 1.9, 95% CI 1.4-2.6) for one mutation vs 12.3 years for no mutations. The results were validated in the Mayo cohort and prognostic significance in both cohorts was independent of International Prognostic Scoring System (IPSS; HR 2.4, 95% CI 1.6-3.6) and dynamic IPSS (DIPSS)-plus (HR 1.9, 95% CI 1.2-3.1), respectively. Two or more mutations were also associated with shortened leukemia-free survival (HR 6.2, 95% CI 3.5-10.7), also Mayo validated. Calreticulin mutations favorably affected survival, independently of both number of mutations and IPSS/DIPSS-plus. We conclude that the 'number' of prognostically detrimental mutations provides added value in the combined molecular and clinical prognostication of PMF.
2014
- miRNA-mRNA integrative analysis in primary myelofibrosis CD34+ cells: role of miR-155/JARID2 axis in abnormal megakaryopoiesis
[Articolo su rivista]
Norfo, Ruggiero; Zini, Roberta; Pennucci, Valentina; Bianchi, Elisa; Salati, Simona; Guglielmelli, Paola; Bogani, Costanza; Fanelli, Tiziana; Mannarelli, Carmela; Rosti, Vittorio; Pietra, Daniela; Salmoiraghi, Silvia; Bisognin, Andrea; Ruberti, Samantha; Rontauroli, Sebastiano; Sacchi, Giorgia; Prudente, Zelia; Barosi, Giovanni; Cazzola, Mario; Rambaldi, Alessandro; Bortoluzzi, Stefania; Ferrari, Sergio; Tagliafico, Enrico; Vannucchi, Alessandro M; Manfredini, Rossella; Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative, Investigators
abstract
Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by megakaryocyte (MK) hyperplasia, bone marrow fibrosis, and abnormal stem cell trafficking. PMF may be associated with somatic mutations in JAK2, MPL, or CALR. Previous studies have shown that abnormal MKs play a central role in the pathophysiology of PMF. In this work, we studied both gene and microRNA (miRNA) expression profiles in CD34(+) cells from PMF patients. We identified several biomarkers and putative molecular targets such as FGR, LCN2, and OLFM4. By means of miRNA-gene expression integrative analysis, we found different regulatory networks involved in the dysregulation of transcriptional control and chromatin remodeling. In particular, we identified a network gathering several miRNAs with oncogenic potential (eg, miR-155-5p) and targeted genes whose abnormal function has been previously associated with myeloid neoplasms, including JARID2, NR4A3, CDC42, and HMGB3. Because the validation of miRNA-target interactions unveiled JARID2/miR-155-5p as the strongest relationship in the network, we studied the function of this axis in normal and PMF CD34(+) cells. We showed that JARID2 downregulation mediated by miR-155-5p overexpression leads to increased in vitro formation of CD41(+) MK precursors. These findings suggest that overexpression of miR-155-5p and the resulting downregulation of JARID2 may contribute to MK hyperplasia in PMF.
2013
- Co-culture of hematopoietic stem/progenitor cells with human osteblasts favours mono/macrophage differentiation at the expense of the erythroid lineage
[Articolo su rivista]
Salati, Simona; Lisignoli, G; Manfredini, C; Pennucci, Valentina; Zini, Roberta; Bianchi, Elisa; Norfo, Ruggiero; Facchini, A; Ferrari, Sergio; Manfredini, Rossella
abstract
Hematopoietic stem cells (HSCs) are located in the bone marrow in a specific microenvironment referred as the hematopoietic stem cell niche, where HSCs interact with a variety of stromal cells. Though several components of the stem cell niche have been identified, the regulatory mechanisms through which such components regulate the stem cell fate are still unknown. In order to address this issue, we investigated how osteoblasts (OBs) can affect the molecular and functional phenotype of Hematopoietic Stem/Progenitor Cells (HSPCs) and vice versa. For this purpose, human CD34+ cells were cultured in direct contact with primary human OBs. Our data showed that CD34+ cells cultured with OBs give rise to higher total cell numbers, produce more CFUs and maintain a higher percentage of CD34+CD38- cells compared to control culture. Moreover, clonogenic assay and long-term culture results showed that co-culture with OBs induces a strong increase in mono/macrophage precursors coupled to a decrease in the erythroid ones. Finally, gene expression profiling (GEP) allowed us to study which signalling pathways were activated in the hematopoietic cell fraction and in the stromal cell compartment after coculture. Such analysis allowed us to identify several cytokine-receptor networks, such as WNT pathway, and transcription factors, as TWIST1 and FOXC1, that could be activated by co-culture with OBs and could be responsible for the biological effects reported above. Altogether our results indicate that OBs are able to affect HPSCs on 2 different levels: on one side, they increase the immature progenitor pool in vitro, on the other side, they favor the expansion of the mono/macrophage precursors at the expense of the erythroid lineage.
2013
- Extracellular purines promote the differentiation of human bone marrow-derived mesenchymal stem cells to the osteogenic and adipogenic lineages
[Articolo su rivista]
Ciciarello, M; Zini, Roberta; Rossi, L; Salvestrini, V; Ferrari, D; Manfredini, Rossella; Lemoli, Rm
abstract
Extracellular nucleotides are potent signaling molecules mediating cell-specific biological functions, mostly within the processes of tissue damage and repair and flogosis. We previously demonstrated that adenosine 5'-triphosphate (ATP) inhibits the proliferation of human bone marrow-derived mesenchymal stem cells (BM-hMSCs), while stimulating, in vitro and in vivo, their migration. Here, we investigated the effects of ATP on BM-hMSC differentiation capacity. Molecular analysis showed that ATP treatment modulated the expression of several genes governing adipogenic and osteoblastic (i.e., WNT-pathway-related genes) differentiation of MSCs. Functional studies demonstrated that ATP, under specific culture conditions, stimulated adipogenesis by significantly increasing the lipid accumulation and the expression levels of the adipogenic master gene PPARγ (peroxisome proliferator-activated receptor-gamma). In addition, ATP stimulated osteogenic differentiation by promoting mineralization and expression of the osteoblast-related gene RUNX2 (runt-related transcription factor 2). Furthermore, we demonstrated that ATP stimulated adipogenesis via its triphosphate form, while osteogenic differentiation was induced by the nucleoside adenosine, resulting from ATP degradation induced by CD39 and CD73 ectonucleotidases expressed on the MSC membrane. The pharmacological profile of P2 purinergic receptors (P2Rs) suggests that adipogenic differentiation is mainly mediated by the engagement of P2Y1 and P2Y4 receptors, while stimulation of the P1R adenosine-specific subtype A2B is involved in adenosine-induced osteogenic differentiation. Thus, we provide new insights into molecular regulation of MSC differentiation.
2013
- Impact Of Prognostically Detrimental Mutations (ASXL1, EZH2, SRSF2, IDH1/2) On Outcomes In Patients With Myelofibrosis Treated With Ruxolitinib In COMFORT-II
[Abstract in Rivista]
Guglielmelli, Paola; Biamonte, Flavia; Pieri, Lisa; Rotunno, Giada; Paoli, Chiara; Fjerza, Rajmonda; Tagliafico, Enrico; Manfredini, Rossella; Artusi, Valentina; Tenedini, Elena; Artuso, Lucia; Bernardis, Isabella; Stalbovskaya, Viktoriya; Squires, Matthew; Harrison, Claire N; Vannucchi, Alessandro M.
abstract
Abstract
Background Ruxolitinib (RUX) is a JAK1 & JAK2 inhibitor that resulted in rapid and durable reductions in splenomegaly and improved disease-related symptoms and quality of life in patients (pts) with myelofibrosis (MF) compared with either placebo (COMFORT-I) or best available therapy (BAT; COMFORT-II). In addition, RUX-treated pts had longer overall survival (OS) compared with placebo and BAT. We recently reported that, among 879 primary MF pts receiving conventional BAT, those harboring a mutation in any one of EZH2, ASXL1, IDH1/2 and SRSF2 constituted an IPSS- and DIPSS-plus prognostic score–independent “high molecular risk” (HMR+) category associated with shorter OS and greater risk of leukemia compared with pts with no mutations (“low molecular risk”; LMR) (Vannucchi AM, et al. Leukemia. 2013). The aim of this study was to analyze the impact of mutational status on spleen volume reduction, anemia development, and OS in pts receiving RUX in the COMFORT II trial.
Patients and methods In COMFORT-II, pts with primary or post–polycythemia vera/–essential thrombocythemia MF were randomized to receive RUX (n=146) or BAT (n=73). Mutations in 12 genes (JAK2, MPL, EZH2, ASXL1, TET2, IDH1/2, CBL, SRSF2, SOCS1, SOCS2, SOCS3, and SH2B3) were genotyped, in DNA derived from whole blood, at baseline in 166 pts (RUX n=120, BAT n=46). Analysis was performed by next-generation sequencing with the Ion Torrent PGM or Roche 454 platform. Sequencing data were analyzed with Nextgene software or Roche 454 Analysis software v2.6 with variant frequency cutoff adjusted to 5%. All mutations were confirmed at least twice. Missense, nonsense, and frameshift mutations only were considered; in the case of novel mutations, SNPs were excluded by database searching and by germline DNA genotyping when available. Development of anemia was defined as a drop of hemoglobin level by more than 1 g/dL from baseline to a value <10 g/dL within the first 48 weeks of treatment. Survival estimates were obtained with Kaplan-Meier method. The treatment effect and the prognostic value of the molecular variables with regard to OS were analyzed by Cox regression and adjusted for the IPSS category.
Results The frequency of mutations was: JAK2V617F 75.47%; MPLW515 7.74%; ASXL1 32.53%; TET2 10.69%; EZH2 7.24%; CBL 4.4%; SRSF2 3.01%; SH2B3 1.3%; IDH1-2 0.7%; SOCS1 0.65%; SOCS2 0.65%; SOCS3 0.0%, with no difference between RUX and BAT. Forty-six (38.3%) and 20 (43.5%) pts in the RUX and BAT groups, respectively, were classified as HMR+.
We first determined whether an HMR+ status impacted the achievement of a ≥35% spleen volume reduction (primary study endpoint). The percentage of RUX treated pts achieving ≥35% spleen volume reduction was 36.3% (16/44) and 33.8% (21/62) at 24 wk and 30.7% (12/39) and 36.3% (20/55) at 48 wk in the HMR+ and LMR categories, respectively. Mean spleen volume reduction was also similar: -29.0% and -23.5% in HMR+ vs -29.9% and -30.6% in LMR pts at 24 and 48 wk. None of the other mutations analyzed correlated with spleen volume reduction in pts receiving RUX.
We also found that an HMR+ status did not predict for the development of anemia associated with RUX administration: the percentage of anemic pts was 74% in the HMR+ group vs 72% in the LMR group. This was also independent of the presence of mutation in any one of the genes associated with JAK2/STAT signaling (JAK2, MPL, SH3B2, CBL, and SOCSs): anemic pts were 74% in mutated vs 72% in wild-type ones.
The survival estimate at 114 wk of follow-up in BAT pts was 0.58 and 0.71 in HMR+ and LMR pts, confirming the negative impact of the mutational risk category. In the RUX arm, the survival estimate was 0.79 and 0.85 for HMR+ and LMR pts, indicating a benefit of RUX treatment in both groups. In the multivariate Cox model, a risk of death with RUX compared with BAT was reduced by 43% (HR=0.57, 95% CI: 0.30-1.08) and LMR patients had
2013
- Integrative Analysis Of mRNA/miRNA Expression Profiles Identified JARID2 As a Shared Target Of Deregulated Mirnas In Primary Myelofibrosis
[Abstract in Rivista]
Zini, Roberta; Norfo, Ruggiero; Pennucci, Valentina; Bianchi, Elisa; Salati, Simona; Paola, Guglielmelli; Andrea, Bisognin; Vittorio, Rosti; Daniela, Pietra; Silvia, Salmoiraghi; Costanza, Bogani; Tiziana, Fanelli; Ruberti, Samantha; Sacchi, Giorgia; Prudente, Zelia; Giovanni, Barosi; Mario, Cazzola; Alessandro, Rambaldi; Stefania, Bortoluzzi; Ferrari, Sergio; Tagliafico, Enrico; Alessandro M., Vannucchi; Manfredini, Rossella
abstract
Ph-negative myeloproliferative neoplasms (MPNs) are characterized by many somatic mutations which have already been shown useful in the prognostic assessment of MPN patients [A.M. Vannucchi et al., Leukemia, 2013]. Moreover, aberrant microRNA (miRNA) expression seems to add to the molecular complexity of MPNs, as specific miRNA signatures capable of discriminating MPN cells from those of normal donors were previously reported [P. Guglielmelli et al., Exp Hematol, 2007]. In order to have a comprehensive picture of miRNA deregulation and its relationship with differential gene expression in primary myelofibrosis (PMF) cells, we obtained gene- (GEP) and miRNA expression profiles (miEP) of CD34+ cells from 31 healthy donors and 42 PMF patients using Affymetrix technology (HG-U219 and miRNA 2.0 arrays). Among 726 differentially expressed genes (DEG) we found that several putative cancer markers (WT1, ANGPT1) and several genes related to PMF progression, i.e. involved in megakaryocyte (MK) differentiation (NFE2, CD9), and fibrosis development (DLK1, LEPR1), were significantly more expressed in PMF samples than in the normal counterpart. Similarly, as regards the miEP, among 74 human differentially expressed miRNAs (DEM) in PMF compared to controls we found the upregulation of several miRNAs associated with hematological malignancies or known as oncomiRs (i.e. hsa-miR-155-5p [S. Jiang et al., Cancer Res, 2010], miRNAs belonging to the miR-17-92 cluster [L. Venturini et al., Blood, 2007]), and other aberrantly expressed miRNAs never described in hematopoiesis (i.e. hsa-miR-335-5p).
Then, in order to construct regulatory networks of the functional human miRNA-target interactions, we performed an integrative analysis (IA) with Ingenuity Pathway analysis software, which combines the miRNA expression profile with computational predicted targets and with the gene expression data. IA between DEG and DEM disclosed a high number of predicted targets with anti-correlated expression to the trend of their targeting miRNAs. Of note, IA identified an interaction network (see Figure) in which the upregulated oncomirs miR-155-5p [R.M. O'Connel et al., J Exp Med, 2008], miR29a-3p [Y.C. Han et al., J Exp Med, 2010] and miR-19b-3p [K.J. Mavrakis et al., Nat Cell Biol, 2010] could explain the downregulation of targets whose lower expression was already described as involved in myeloproliferative phenotypes, such as NR4A3, CDC42, HMGB3. Additionally, IA disclosed the chromatin remodeler JARID2, which is frequently deleted in leukemic transformation of chronic myeloid malignancies, as a shared target of several upregulated miRNAs in PMF samples (i.e. miR-155-5p, miR-152-3p). Noteworthy, these miRNA-mRNA interactions were functionally confirmed by 3' UTR luciferase reporter assays.
Next, in order to characterize the role of JARID2 in PMF pathogenesis, we performed RNAi-mediated gene silencing experiments on CD34+ cells of healthy donor. Interestingly, inhibition of JARID2 expression produces in silenced cells a significant increase of CD41 expression when compared with control (28.6±3.1% vs 15.3±1.8% at day 8, 52.6±7.6% vs 35.4±4.9% at day 12 of serum free liquid culture) and a remarkable increase in CFU-MK colonies (59.6±6.5% vs 39.8±5.9%). The values are reported as mean ± 2S.E.M from five independent experiments.
Moreover, morphological analysis after May-Grunwald-Giemsa staining showed that JARID2 silencing induces in normal CD34+ cells a considerable enrichment in MK precursors at different stages of maturation.
This study allowed the identification of different networks possibly involved in PMF onset, highlighting the potential contribution of miRNAs to PMF pathogenesis. Furthermore, for the first time, we demonstrated that the JARID2 downregulation in CD34+ cells might contribute to the abnormal megakaryopoiesis typical of PMF.
2013
- Isolation of human keratinocyte stem cells and high-throughput screening approach for their characterization
[Abstract in Atti di Convegno]
Di Rocco, Antonio; Carulli, Sonia; Tenedini, Elena; Bianchi, Elisa; Tagliafico, Enrico; Manfredini, Rossella; Pellegrini, Graziella; DE LUCA, Michele
abstract
In the last three decades, regenerative medicine has opened new horizons for the in vitro
reconstruction of epithelial tissues and gene therapy treatment of skin disorders involving the
use of adult keratinocyte stem cells (KSCs). Although the ability to identify and isolate these cells
represents an important prerequisite for the development of these approaches, molecular markers
and their precise in vivo localization are still lacking. In order to define genes involved in the control
of stemness and commitment of KSCs, we developed a non-invasive, stem cell-preserving magnetic
micro beads based method in order to obtain a KSCs enriched population for high throughput
screening experiments. After 3T3 murine fibroblast feeder layer depletion from our keratinocyte
cultures, we isolated a subpopulation of basal epithelial cells on the basis of the different expression
levels of the a6β4 integrin. By using different approaches, including clonal analysis and p63 bright cells
quantification, we clearly showed that a6β4 integrin bright cells have greater growth potential and
clonogenic capacity compared to the remaining cell fraction and they include the KSCs population.
Comparing gene expression profile of a KSCs-enriched and a terminally differentiated cell population
coming from the same original primary cell culture we defined a set of genes most probably involved
in stemness maintenance. Ongoing gene profiling on single clone type will allow us to validate this
gene signature and to start functional studies on selected genes. Extending this approach to different
ectodermal derived tissues will provide a genome wide signature of the molecular pathways underlying
self-renewal, commitment and differentiation of KSCs.
2013
- Mutations and prognosis in primary myelofibrosis
[Articolo su rivista]
Vannucchi, Am; Lasho, Tl; Guglielmelli, P; Biamonte, F; Pardanani, A; Pereira, A; Finke, C; Score, J; Gangat, N; Mannarelli, C; Ketterling, Rp; Rotunno, G; Knudson, Ra; Susini, Mc; Laborde, Rr; Spolverini, A; Pancrazzi, A; Pieri, L; Manfredini, Rossella; Tagliafico, Enrico; Zini, Roberta; Jones, A; Zoi, K; Reiter, A; Duncombe, A; Pietra, D; Rumi, E; Cervantes, F; Barosi, G; Cazzola, M; Cross, Nc; Tefferi, A.
abstract
Patient outcome in primary myelofibrosis (PMF) is significantly influenced by karyotype. We studied 879 PMF patients to determine the individual and combinatorial prognostic relevance of somatic mutations. Analysis was performed in 483 European patients and the seminal observations were validated in 396 Mayo Clinic patients. Samples from the European cohort, collected at time of diagnosis, were analyzed for mutations in ASXL1, SRSF2, EZH2, TET2, DNMT3A, CBL, IDH1, IDH2, MPL and JAK2. Of these, ASXL1, SRSF2 and EZH2 mutations inter-independently predicted shortened survival. However, only ASXL1 mutations (HR: 2.02; P<0.001) remained significant in the context of the International Prognostic Scoring System (IPSS). These observations were validated in the Mayo Clinic cohort where mutation and survival analyses were performed from time of referral. ASXL1, SRSF2 and EZH2 mutations were independently associated with poor survival, but only ASXL1 mutations held their prognostic relevance (HR: 1.4; P=0.04) independent of the Dynamic IPSS (DIPSS)-plus model, which incorporates cytogenetic risk. In the European cohort, leukemia-free survival was negatively affected by IDH1/2, SRSF2 and ASXL1 mutations and in the Mayo cohort by IDH1 and SRSF2 mutations. Mutational profiling for ASXL1, EZH2, SRSF2 and IDH identifies PMF patients who are at risk for premature death or leukemic transformation.
2012
- Characterisation and discovery of novel miRNAs and moRNAs in JAK2V617F mutated SET2 cells
[Articolo su rivista]
Bortoluzzi, S; Bisognin, A; Biasolo, M; Guglielmelli, P; Biamonte, F; Norfo, Ruggiero; Manfredini, Rossella; Vannucchi, A. M.
abstract
In order to gain insights into a possible role of microRNAs in myeloproliferative neoplasms, we performed short RNAs massive sequencing and extensive bioinformatic analysis in the JAK2V617F mutated SET2 cell line. Overall, 652 known mature miRNAs were detected, of which 21 were highly expressed, thus being responsible of most of miRNA-mediated gene repression. microRNA putative targets were enriched in specific signaling pathways, providing information about cell activities under massive post-transcriptional regulation. The majority of miRNAs were mixtures of sequence variants, called isomiRs, mainly due to alternative, non-canonical processing of hairpin precursors. We also identified 78 novel miRNAs (miRNA*) derived from known hairpin precursors. Both major and minor (*) form of miRNAs were expressed concurrently from half of expressed hairpins, highlighting the relevance of miRNA* and the complexity of strand selection bias regulation. Finally, we discovered that SET2 cells express a number of miRNA-offset RNAs (moRNAs), short RNAs derived from genomic regions flanking mature miRNAs. We provide novel data about the possible origin of moRNAs, while their functional role remains to be elucidated. Overall, this study shed light on the complexity of microRNA-mediated gene regulation in SET2 cells and represents the basis for future studies in JAK2V617F mutated cellular models.
2012
- Purinergic signaling inhibits human acute myeloblastic leukemia cell proliferation, migration, and engraftment in immunodeficient mice
[Articolo su rivista]
Salvestrini, V; Zini, Roberta; Rossi, L; Gulinelli, S; Manfredini, Rossella; Bianchi, Elisa; Piacibello, W; Caione, L; Migliardi, G; Ricciardi, Mr; Tafuri, A; Romano, M; Salati, Simona; Di Virgilio, F; Ferrari, Sergio; Baccarani, M; Ferrari, D; Lemoli, R. M.
abstract
Extracellular ATP and UTP nucleotides increase the proliferation and engraftment potential of normal human hematopoietic stem cells via the engagement of purinergic receptors (P2Rs). In the present study, we show that ATP and UTP have strikingly opposite effects on human acute myeloblastic leukemia (AML) cells. Leukemic cells express P2Rs. ATP-stimulated leukemic cells, but not normal CD34+ cells, undergo down-regulation of genes involved in cell proliferation and migration, whereas cell-cycle inhibitors are up-regulated. Functionally, ATP induced the inhibition of proliferation and accumulation of AML cells, but not of normal cells, in the G0 phase of the cell cycle. Exposure to ATP or UTP inhibited AML-cell migration in vitro. In vivo, xenotransplantation experiments demonstrated that the homing and engraftment capacity of AML blasts and CD34+CD38- cells to immunodeficient mice BM was significantly inhibited by pretreatment with nucleotides. P2R-expression analysis and pharmacologic profiling suggested that the inhibition of proliferation by ATP was mediated by the down-regulation of the P2X7R, which is up-regulated on untreated blasts, whereas the inhibition of chemotaxis was mainly mediated via P2Y2R and P2Y4R subtypes. We conclude that, unlike normal cells, P2R signaling inhibits leukemic cells and therefore its pharmacologic modulation may represent a novel therapeutic strategy.
2012
- Valproic acid triggers erythro/megakaryocyte lineage decision through induction of GFI1B and MLLT3 expression
[Articolo su rivista]
Zini, Roberta; Norfo, Ruggiero; Ferrari, Francesco; Bianchi, Elisa; Salati, Simona; Pennucci, Valentina; Sacchi, Giorgia; Carboni, Chiara; Ceccherelli, Gb; Tagliafico, Enrico; Ferrari, Sergio; Manfredini, Rossella
abstract
Histone deacetylase inhibitors represent a family of targeted anticancer compounds that are widely used against hematological malignancies. So far little is known about their effects on normal myelopoiesis. Therefore, in order to investigate the effect of histone deacetylase inhibitors on the myeloid commitment of hematopoietic stem/progenitor cells, we treated CD34(+) cells with valproic acid (VPA). Our results demonstrate that VPA treatment induces H4 histone acetylation and hampers cell cycle progression in CD34(+) cells sustaining high levels of CD34 protein expression. In addition, our data show that VPA treatment promotes erythrocyte and megakaryocyte differentiation. In fact, we demonstrate that VPA treatment is able to induce the expression of growth factor-independent protein 1B (GFI1B) and of mixed-lineage leukemia translocated to chromosome 3 protein (MLLT3), which are crucial regulators of erythrocyte and megakaryocyte differentiation, and that the up-regulation of these genes is mediated by the histone hyperacetylation at their promoter sites. Finally, we show that GFI1B inhibition impairs erythroid and megakaryocyte differentiation induced by VPA, while MLLT3 silencing inhibits megakaryocyte commitment only. As a whole, our data suggest that VPA sustains the expression of stemness-related markers in hematopoietic stem/progenitor cells and is able to interfere with hematopoietic lineage commitment by enhancing erythrocyte and megakaryocyte differentiation and by inhibiting the granulocyte and mono-macrophage maturation.
2011
- Purinergic stimulation of human mesenchymal stem cells potentiates their chemotactic response to CXCL12 and increases the homing capacity and production of proinflammatory cytokines
[Articolo su rivista]
Ferrari, D; Gulinelli, S; Salvestrini, V; Lucchetti, G; Zini, Roberta; Manfredini, Rossella; Caione, L; Piacibello, W; Ciciarello, M; Rossi, L; Idzko, M; Ferrari, Sergio; Di Virgilio, F; Lemoli, Rm
abstract
Objective: Extracellular adenosine triphosphate (ATP) is a well-recognized mediator of cell-to-cell communication. Here we show ATP effects on bone marrow (BM)-derived human mesenchymal stem cell (hMSCs) functions. Materials and Methods: ATP-induced modification of hMSCs gene expression profile was assessed by Affymetrix technology. Clonogenic and migration assays in vitro, as well as xenotransplant experiments in vivo, were performed to evaluate the effects of ATP on hMSCs proliferation and BM homing. Enzyme-linked immunosorbent assays were used to assess hMSCs cytokines production, whereas T-cell cultures demonstrated the immunoregulatory activity of ATP-treated hMSCs. Results: hMSCs were resistant to the cytotoxic effects of ATP, as demonstrated by the lack of morphological and mitochondrial changes or release of intracellular markers of cell death. Gene expression profiling revealed that ATP-stimulated hMSCs underwent a downregulation of genes involved in cell proliferation, whereas those involved in cell migration were strongly upregulated. The inhibitory activity of ATP on hMSCs proliferation was confirmed by assessing clonogenic stromal progenitors. ATP potentiated the chemotactic response of hMSCs to the chemokine CXCL12, and increased their spontaneous migration. In vivo, the homing capacity of hMSCs to the BM of immunodeficient mice was significantly increased by pretreatment with ATP. Moreover, ATP increased the production of the proinflammatory cytokines interleukin-2, interferon-γ, and interleukin-12p70, while decreasing the anti-inflammatory cytokine interleukin-10, and this finding was associated with the reduced ability of MSCs to inhibit T-cell proliferation. Conclusions: Our data show that purinergic signaling modulates hMSCs functions and highlights a role for extracellular nucleotides in hMSCs biology.
2010
- Bone marrow-derived hematopoietic cells undergo myogenic differentiation following a Pax-7 independent pathway
[Articolo su rivista]
Xynos, A; Corbella, P; Belmonte, N; Zini, Roberta; Manfredini, Rossella; Ferrari, G.
abstract
Several reports showed that hematopoietic stem cells (HSCs) participate in muscle regeneration, raising hope for their therapeutic potential for degenerative muscle diseases. However, proof that HSCs are able to reprogram their fate and enter a myogenic pathway, remains elusive. We demonstrate that murine bone marrow (BM)-derived hematopoietic cells, carrying reporter genes controlled by muscle-specific regulatory elements from the Myf5, myosin light chain (MLC3F), or MCK genes, are induced by myoblasts to activate muscle-specific genes. This potential resides in the more undifferentiated progenitors, expressing surface markers typical of HSCs. Comparative gene expression profiling of CD45(+)/Sca1(+) cells isolated from muscle or BM shows that hematopoietic cells participate to muscle regeneration, by undergoing a profound although incomplete myogenic reprogramming on interaction with the muscle microenviroment. These cells undergo specification and differentiation independently from Pax7 and MyoD, and lack Pax7-associated properties, such as self-renewal and proliferation, distinguishing from satellite cells. Our findings indicate that hematopoietic cells, on seeding in the muscle, become a distinct cell population endowed with myogenic potential.
2010
- c-Myb supports erythropoiesis by transactivating KLF1 and LMO2 expression
[Abstract in Atti di Convegno]
Bianchi, Elisa; Zini, Roberta; Salati, Simona; Tenedini, Elena; Norfo, Ruggiero; Ferrari, Sergio; Manfredini, Rossella
abstract
The c-Myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during
differentiation. c-myb is essential for the hematopoietic development, as c-myb-/- mice die at E15 due to failure of fetal
hepatic erythropoiesis. To gain further insights into the role of c-myb during the hematopoietic lineage commitment, we
studied the effects of c-Myb silencing in human CD34+ hematopoietic stem/progenitor cells. c-Myb silencing in CD34+
cells was performed by transfection of siRNAs using the Amaxa Nucleofector® Technology. In order to keep c-Myb
expression silenced for all the commitment phase of CD34+ cells, each sample was nucleofected 3 times, once a day.
Moreover, to exclude non-specific effects of siRNA nucleofection, for each experiment, together with the sample
transfected with the siRNAs targeting c-Myb, one sample electroporated without siRNAs and one transfected with a
non-targeting siRNA were performed. c-Myb silencing effects on CD34+ cells differentiation ability were studied by
methylcellulose and collagen-based clonogenic assays and by morphological and immunophenotypic analyses after
liquid culture. Furthermore, we investigated by microarray analysis the changes in gene expression induced by c-Myb
silencing. Methylcellulose assay revealed a remarkable increase of the percentage of monocyte (CFU-M) colonies and a
decrease of the erythroid ones (BFU-E) in c-Myb-silenced CD34+ cells. Moreover, collagen-based clonogenic assay
demonstrated that c-Myb silencing strongly enhances the megakaryocyte commitment of CD34+ cells. In agreement
with these data, flow cytometric analysis showed an increase in mono-macrophage and megakaryocyte fractions in cmyb-silenced
cells, while the erythroid population was strongly decreased. Morphological evaluation of May
Grunwald-Giemsa stained cytospins further supported the conclusion that c-myb silencing forces the CD34+ cells
commitment towards the macrophage and megakaryocyte lineages at the expense of the erythroid one. Gene expression
profiling of c-Myb silenced CD34+ cells enabled us to identify new putative targets which can account for c-Myb
knockdown effects. Indeed, Chromatin Immunoprecipitation and Luciferase reporter assay demonstrated that c-Myb
binds to KLF1 and LMO2 promoters and transactivates their expression. Functional rescue experiments showed that the
retroviral vector-mediated overexpression of KLF1 and LMO2 transcription factors in c-Myb silenced cells is able to
rescue, at least in part, the impaired erythroid differentiation. Our data collectively demonstrate that c-Myb plays a
pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at
the expense of megakaryocyte diffentiation. In particular, we identified c-Myb-driven KLF1 and LMO2 transactivation
as the molecular mechanism through which c-Myb regulates erythroid versus megakaryocyte lineage fate decision.
2010
- c-Myb supports erythropoiesis through the transactivation of KLF1 and LMO2 expression.
[Articolo su rivista]
Bianchi, Elisa; Zini, Roberta; Salati, Simona; Tenedini, Elena; Norfo, Ruggiero; Tagliafico, Enrico; Manfredini, Rossella; Ferrari, Sergio
abstract
The c-Myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-Myb in human CD34+ hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity towards the macrophage and megakaryocyte lineages, while erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphological and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-Myb-silenced CD34+ cells identified the transcription factors KLF1 and LMO2 as putative targets which can account for c-Myb knockdown effects. Indeed, Chromatin Immunoprecipitation and Luciferase reporter assay demonstrated that c-Myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently, the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-Myb silencing, while only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34+ cells. Our data collectively demonstrate that c-Myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed, we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision.
2009
- Mechanistic insight into WEB-2170-induced apoptosis in human acute myelogenous leukemia cells. the crucial role of PTEN.
[Articolo su rivista]
Cellai, C; Laurenzana, A; Bianchi, Elisa; Sdelci, S; Manfredini, Rossella; Vannucchi, Am; Caporale, R; Balliu, M; Mannelli, F; Ferrari, Sergio; Bosi, A; Miniati, D; Cocco, Pl; Veronneau, S; Stankova, J; Paoletti, F.
abstract
OBJECTIVE:This study aimed to investigate the mechanisms of action of WEB-2170, an inverse agonist of platelet-activating factor receptor, capable of inducing apoptosis in human acute myelogenous leukemia (AML) cells.MATERIAL AND METHODS:Gene expression profiling followed by cytofluorimetric, morphologic, and biologic analyses were used to monitor WEB-2170 effects in AML cell lines (ie, NB4, KG1, NB4-MR4, THP1, and U937) and blasts from patients with different AML (M0-M5) subtypes. PTEN silencing with small interfering RNA was also performed.RESULTS:We have demonstrated that drug-mediated cytostasis/apoptosis in NB4 cells is characterized by upregulation of cyclin G2, p21/WAF1, NIX, TNF-alpha, and PTEN expression, and downregulation of cyclin D2 and BCL2 expression. We observed an increase in PTEN protein accompanied by a decrease in phospho-extracellular signal-regulated kinase 2 (ERK2) and phospho-AKT, and by forkhead box O3a (FOXO3a) cytoplasmic-nuclear translocation; the mitochondrial cytochrome C release and PARP cleavage marked the late apoptotic steps. We have found that WEB-2170 triggered apoptosis in NB4, KG1, and NB4-MR4 cells where PTEN was expressed, but not in THP1 and U937 cells where PTEN was absent. Finally, we show that PTEN silencing in NB4 cells by PTEN-specific small interfering RNA resulted in a significant reduction of drug-induced apoptosis.CONCLUSION:We demonstrated that WEB-2170 is a powerful antileukemic agent with interesting translational opportunities to treat AML and described mechanisms of drug-induced intrinsic and extrinsic apoptosis both in AML cell lines and blasts from AML patients by addressing PTEN as the master regulator of the whole process.
2009
- Molecular and functional analysis of the stem cell compartment of chronic myelogenous leukemia reveals the presence of a CD34- cell population with intrinsic resistance to imatinib
[Articolo su rivista]
Lemoli, Rm; Salvestrini, V; Bianchi, Elisa; Bertolini, F; Fogli, M; Amabile, M; Tafuri, A; Salati, Simona; Zini, Roberta; Testoni, N; Rabascio, C; Rossi, L; Martin Padura, I; Castagnetti, F; Marighetti, P; Martinelli, G; Baccarani, M; Ferrari, Sergio; Manfredini, Rossella
abstract
We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin–CD34–) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular karyotyping and quantitative analysis of BCR-ABL transcript demonstrated that approximately one-third of CD34– cells are leukemic. CML Lin–CD34– cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells and cell cycling, and increased clonogenic activity and expression of BCR-ABL transcript. Lin–CD34– cells showed hematopoietic cell engraftment rate in 2 immunodeficient mouse strains similar to Lin-CD34+ cells, whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell-cycle arrest genes and genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated compared with normal counterparts. Phenotypic analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin–CD34– cells. Imatinib mesylate did not reduce fusion transcript levels, BCR-ABL kinase activity, and clonogenic efficiency of CML Lin–CD34– cells in vitro. Moreover, leukemic CD34– cells survived exposure to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34– leukemic stem cell subset in CML with peculiar molecular and functional characteristics.
2009
- Molecular profile of CD34+ stem/progenitor cells according to JAK2V617F mutation status in essential thrombocythemia.
[Articolo su rivista]
Catani, L; Zini, Roberta; Sollazzo, D; Ottaviani, E; Vannucchi, Am; Ferrari, Sergio; Baccarani, M; Vianelli, N; Lemoli, Rm; Manfredini, Rossella
abstract
Essential thrombocythemia (ET) is mainly characterized by the abnormal proliferation of a malignant megakaryocytic clone and by persistent thrombocytosis. Recently, a JAK2 mutation (JAK2V617F) has been reported in ET and other myeloproliferative neoplasms.
2008
- Isolation and characterization of murine liver resident stem cell
[Articolo su rivista]
Conigliaro, A; Colletti, M; Cicchini, C; Guerra, Mt; Manfredini, Rossella; Zini, Roberta; Bordoni, V; Siepi, F; Leopizzi, M; Tripodi, M; Amicone, L.
abstract
Increasing evidence provides support that mammalian liver contains stem/progenitor cells, but their molecular phenotype, embryological derivation, biology and their role in liver cell turnover and regeneration remain to be further clarified. In this study, we report the isolation, characterization and reproducible establishment in line of a resident liver stem cell (RLSC) with immunophenotype and differentiative potentiality distinct from other previously described liver precursor/stem cells. RLSCs, derived from fetal and neonatal murine livers as well as from immortalized hepatocytic MMH lines and established in lines, are Sca+, CD34-, CD45-, -fetoprotein+ and albumin-. This molecular phenotype suggests a non-hematopoietic origin. RLSC transcriptional profile, defined by microArray technology, highlighted the expression of a broad spectrum of 'plasticity-related genes' and 'developmental genes' suggesting a multi-differentiative potentiality. Indeed, RLSCs spontaneously differentiate into hepatocytes and cholangiocytes and, when cultured in appropriate conditions, into mesenchymal and neuro-ectodermal cell lineages such as osteoblasts/osteocytes, chondrocytes, astrocytes and neural cells. RLSC capability to spontaneously differentiate into hepatocytes, the lack of albumin expression and the broad differentiative potentiality locate them in a pre-hepatoblast/liver precursor cells hierarchical position. In conclusion, RLSCs may provide a useful tool to improve liver stem cell knowledge and to assess new therapeutic approaches for liver diseases.
2008
- Role of CD34 antigen in myeloid differentiation of human hematopoietic progenitor cells
[Articolo su rivista]
Salati, Simona; Zini, Roberta; Bianchi, Elisa; Testa, Anna; Mavilio, Fulvio; Manfredini, Rossella; Ferrari, Sergio
abstract
CD34 is a transmembrane protein that is strongly expressed on hematopoietic stem/progenitor cells (HSCs); despite its importance as a marker of HSCs, its function is still poorly understood, although a role in cell adhesion has been demonstrated. To characterize the function of CD34 antigen on human HSCs, we examined, by both inhibition and overexpression, the role of CD34 in the regulation of HSC lineage differentiation. Our results demonstrate that CD34 silencing enhances HSC granulocyte and megakaryocyte differentiation and reduces erythroid maturation. In agreement with these results, the gene expression profile of these cells reveals the upregulation of genes involved in granulocyte and megakaryocyte differentiation and the downregulation of erythroid genes. Consistently, retroviral-mediated CD34 overexpression leads to a remarkable increase in erythroid progenitors and a dramatic decrease in granulocyte progenitors, as evaluated by clonogenic assay. Together, these data indicate that the CD34 molecule promotes the differentiation of CD34+ hematopoietic progenitors toward the erythroid lineage, which is achieved, at least in part, at the expense of granulocyte and megakaryocyte lineages.
2008
- Silencing CD34 antigen in human hematopoietic stem cells
[Articolo su rivista]
Zini, Roberta; Salati, Simona; Bianchi, Elisa; Ferrari, Sergio; Manfredini, Rossella
abstract
CD34 is a highly glycosylated transmembrane protein strongly expressed on hematopoietic stem/progenitor cells (HSPCs); despite its importance as a marker of HSPCs, its function is still poorly understood, even if a role in cell adhesion has been demonstrated. In order to characterize the function of CD34 antigen in human HSPCs, we evaluated by small interfering RNAs (siRNAs) mediated gene silencing the role of CD34 antigen in HSPCs differentiation. By using the Nucleofection Amaxa technology for siRNA transfection in HSPCs, we obtained a rapid and effective down-regulation of the CD34 antigen. In this paper, we have demonstrated that CD34 silencing in HSPCs enhances their granulocyte and megakaryocyte differentiation and reduces erythroid maturation as shown by clonogenic assay, morphological analysis and expression of differentiation markers. In agreement with these results, the gene expression profile of HSPCs-silenced cells reveals the up-regulation of genes involved in granulocyte and megakaryocyte commitment and the down-regulation of erythroid genes. These data indicate that CD34 transmembrane protein promotes the differentiation of CD34+ hematopoietic progenitors towards the erythroid lineage at the expense of granulocyte and megakaryocyte ones.
2007
- Eosinophils, but not neutrophils, exibit an efficient DNA repair machinary and high nucleolar activity
[Articolo su rivista]
Salati, Simona; Bianchi, Elisa; Zini, Roberta; Tenedini, Elena; Quaglino, Daniela; Manfredini, Rossella; Ferrari, Sergio
abstract
BACKGROUND AND OBJECTIVES: Traditionally eosinophils have been considered terminally differentiated cells that play a role in host protection against parasites. However, there is some evidence showing that eosinophils are, in fact, multifunctional leukocytes involved in inflammatory responses, as well as in tissue homeostasis. We characterized the transcriptome profile of human eosinophils, and, for the purpose of comparison, the transcriptome profile of neutrophils, monocytes and hematopoietic progenitor cells. Moreover, we studied the activation of selected cellular processes for which a significant differential expression was demonstrated. DESIGN AND METHODS: We profiled gene expression using Affymetrix GeneChips. DNA repair capacity was tested using the comet assay. Nucleoli and their activity were characterized by transmission electron microscopy analysis, silver staining of nucleolus regions (AgNOR) and RNA staining. RESULTS: Gene expression profiling showed that eosinophils appear hierarchically closer to monocytes than to neutrophils. Gene ontology mapping of differentially expressed genes revealed that eosinophils express categories very similar to those expressed by monocytes, related to DNA repair and nucleolar functions. Moreover, our data show that eosinophils and monocytes maintain the ability to repair both double and single strand DNA breaks, whereas neutrophils lack this capacity. Furthermore, eosinophils exhibit nucleolar activity, which is lacking in neutrophils, but resembles that in monocytes. INTERPRETATION AND CONCLUSIONS: The presence of large, active nucleoli in eosinophils, coupled to marked activity of DNA repair systems, suggests that eosinophils are not terminally differentiated cells. Indeed, their transcriptome profile and functional properties are more similar to those of non-terminally differentiated cells such as monocytes, rather than to neutrophils.
2007
- MicroRNA expression profile in granulocytes from primary myelofibrosis patients
[Articolo su rivista]
Guglielmelli, P; Tozzi, L; Pancrazzi, A; Bogani, C; Antonioli, E; Ponziani, V; Poli, G; Zini, Roberta; Ferrari, Sergio; Manfredini, Rossella; Bosi, A; Vannucchi, Am
abstract
Expression profiling of microRNA (miRNA) was performed in granulocytes isolated from patients with primary myelofibrosis (PMF), with the aim of identifying abnormally expressed miRNAs in comparison with normal subjects or patients with polycythemia vera (PV) or essential thrombocythemia (ET).PATIENTS AND METHODS:Using stem loop-primed reverse transcription and TaqMan quantitative real-time polymerase chain reaction, the expression of 156 mature miRNAs was evaluated using pooled granulocytes from PMF patients, either wild-type or JAK2(617V>F) mutant with >51% allele burden, and control subjects. Differentially expressed miRNAs were then validated on additional control and PMF samples, and also on PV or ET granulocytes.RESULTS:There was a global downregulation of miRNA expression in PMF granulocytes; 60 miRNAs, of 128 called present, displayed differential expression compared to normal samples. Twelve miRNAs, which had been selected based on statistically different expression level, were finally validated. In PMF granulocytes, levels of miR-31, -150, and -95 were significantly lower, while those of miR-190 significantly greater, than control and PV or ET samples; on the other hand, miR-34a, -342, -326, -105, -149, and -147 were similarly reduced in patients with PMF, PV, or ET compared to controls. Increased expression of miR-182 and -183 correlated with JAK2(617V>F) allele burden. Three in silico-predicted putative target genes (DTR, HMGA2, and MYB), showed deregulated expression in PMF granulocytes that correlated with expression level of regulatory miRNA.CONCLUSIONS:A defined miRNA profile distinguishes PMF granulocytes from those of normal subjects and, partially, also from PV or ET patients
2007
- Molecular profiling of CD34+ cells in idiopathic myelofibrosis identifies a set of disease-associated genes and reveals the clinical significance of Wilms' tumor gene 1 (WT1)
[Articolo su rivista]
Guglielmelli, P; Zini, Roberta; Bogani, C; Salati, Simona; Pancrazzi, A; Bianchi, Elisa; Mannelli, F; Ferrari, Sergio; Le Bousse Kerdilès, Mc; Bosi, A; Barosi, G; Migliaccio, Ar; Manfredini, Rossella; Vannucchi, A. M.
abstract
This study was aimed at the characterization of a gene expression signature of the pluripotent hematopoietic CD34(+) stem cell in idiopathic myelofibrosis (IM), which would eventually provide novel pathogenetic insights and/or diagnostic/prognostic information. Aberrantly regulated genes were revealed by transcriptome comparative microarray analysis of normal and IM CD34(+) cells; selected genes were also assayed in granulocytes. One-hundred seventy four differentially expressed genes were identified and in part validated by quantitative polymerase chain reaction. Altered gene expression was corroborated by the detection of abnormally high CD9 or CD164, and low CXCR4, membrane protein expression in IM CD34(+) cells. According to class prediction analysis, a set of eight genes (CD9, GAS2, DLK1, CDH1, WT1, NFE2, HMGA2, and CXCR4) properly recognized IM from normal CD34(+) cells. These genes were aberrantly regulated also in IM granulocytes that could be reliably differentiated from control polycythemia vera and essential thrombocythemia granulocytes in 100% and 81% of cases, respectively. Abnormal expression of HMGA2 and CXCR4 in IM granulocytes was dependent on the presence and the mutational status of JAK2(V617F) mutation. The expression levels of both CD9 and DLK1 were associated with the platelet count, whereas higher WT1 expression levels identified IM patients with more active disease, as revealed by elevated CD34(+) cell count and higher severity score. In conclusion, molecular profiling of IM CD34(+) cells uncovered a limited number of genes with altered expression that, beyond their putative role in disease pathogenesis, are associated with patients' clinical characteristics and may have potential prognostic application.
2007
- The extracellular nucleotide UTP is a potent inducer of hematopoietic stem cell migration
[Articolo su rivista]
Rossi, L; Manfredini, Rossella; Bertolini, F; Ferrari, D; Fogli, M; Zini, Roberta; Salati, Simona; Salvestrini, V; Gulinelli, S; Adinolfi, E; Ferrari, Sergio; Di Virgilio, F; Baccarani, M; Lemoli, R. M.
abstract
Homing and engraftment of hematopoietic stem cells (HSCs) to the bone marrow (BM) involve a complex interplay between chemokines, cytokines, and nonpeptide molecules. Extracellular nucleotides and their cognate P2 receptors are emerging as key factors of inflammation and related chemotactic responses. In this study, we investigated the activity of extracellular adenosine triphosphate (ATP) and uridine triphosphate (UTP) on CXCL12-stimulated CD34+ HSC chemotaxis. In vitro, UTP significantly improved HSC migration, inhibited cell membrane CXCR4 down-regulation by migrating CD34+ cells, and increased cell adhesion to fibronectin. In vivo, preincubation with UTP significantly enhanced the BM homing efficiency of human CD34+ cells in immunodeficient mice. Pertussis toxin blocked CXCL12- and UTP-dependent chemotactic responses, suggesting that G-protein alpha-subunits (Galphai) may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP- and CXCL12-treated CD34+ cells and in vitro inhibition assays demonstrated that Rho guanosine 5'-triphosphatase (GTPase) Rac2 and downstream effectors Rho GTPase-activated kinases 1 and 2 (ROCK1/2) are involved in UTP-promoted/CXCL12-dependent HSC migration. Our data suggest that UTP may physiologically modulate the homing of HSCs to the BM, in concert with CXCL12, via the activation of converging signaling pathways between CXCR4 and P2Y receptors, involving Galphai proteins and RhoGTPases
2007
- Transcriptional profiles in melanocytes from clinically unaffected skin distinguish the neoplastic growth pattern in patients with melanoma
[Articolo su rivista]
Magnoni, Cristina; Tenedini, Elena; Ferrari, Francesco; Benassi, Luisa; Bernardi, Chiara; Gualdi, Giulio; Bertazzoni, Giorgia; Roncaglia, Enrica; Fantoni, Luca Isaia; Manfredini, Rossella; Bicciato, Silvio; Ferrari, Sergio; Giannetti, Alberto; Tagliafico, Enrico
abstract
Background It is generally accepted that sunlight may contribute to the development of melanoma. Objectives To analyse gene expression of melanocytes obtained from clinically unaffected skin of patients with melanoma and healthy controls before and after exposure to ultraviolet B radiation. Methods Using GeneChip array technology, the gene expression of melanocytes obtained from the two donor groups was profiled, in order to identify transcriptional differences affecting susceptibility to melanoma. Results The data collected did not show any difference between the expression profiles of melanocytes purified from normal donors and from patients with melanoma that was able to give a statistically significant class separation. However, by means of unsupervised clustering our data could be divided into two main classes. The first class included the transcriptome profiles of melanocytes obtained from skin samples of patients with a vertical growth phase (VGP) melanoma, while the second class included the transcriptome profiles of melanocytes obtained from skin samples of patients with a radial growth phase (RGP) melanoma. Conclusions These data suggest that melanocytes in patients with VGP and RGP melanomas show significant differences in gene expression profiles, which allow us to classify patients with melanoma also from clinically unaffected skin.
2006
- Hepatocyte growth factor favors monocyte differentiation into regulatory interleukin (IL)-10++IL-12low/neg accessory cells with dendritic-cell features
[Articolo su rivista]
Rutella, S; Bonanno, G; Procoli, A; Mariotti, A; de Ritis, Dg; Curti, A; Danese, S; Pessina, G; Pandolfi, S; Natoni, F; Di Febo, A; Scambia, G; Manfredini, Rossella; Salati, Simona; Ferrari, Sergio; Pierelli, L; Leone, G; Lemoli, R. M.
abstract
Several hematopoietic growth factors, including interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1), promote the differentiation of tolerogenic dendritic cells (DCs). Hepatocyte growth factor (HGF) is a pleiotropic cytokine whose effects on human DC differentiation and function have not been investigated. Monocytes cultured with HGF (HGFMo) differentiated into accessory cells with DC-like morphology, released low amounts of IL-12p70 and up-regulated IL-10 both at the mRNA and at the protein level. Upon activation with HGFMo, allogeneic CD4+CD25- T cells expressed the T regulatory (Treg)-associated transcription factor FoxP3, proliferated poorly, and released high levels of IL-10. Interestingly, blockade of surface immunoglobulin-like transcript 3 (ILT3) on HGFMo or neutralization of secreted IL-10 translated into partial restoration of T-cell proliferation. Secondary stimulation of HGFMo-primed CD4+ T cells with immunogenic DCs differentiated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 from monocytes of the same donor resulted in measurable T-cell proliferation. HGFMo-primed CD4+ T cells significantly inhibited the proliferation of naive CD4+CD25- T cells in a cell-contact-dependent manner. Finally, DNA microarray analysis revealed a unique gene-expression profile of HGF-activated monocytes. Collectively, our findings point to a novel role for HGF in the regulation of monocyte/DC functions that might be exploited therapeutically.
2006
- IDENTIFICATION OF A MOLECULAR SIGNATURE PREDICTIVE OF REFRACTORINESS IN ACUTE MYELOID LEUKEMIA
[Abstract in Atti di Convegno]
Tenedini, Elena; Tagliafico, Enrico; Manfredini, Rossella; Ferrari, Francesco; Roncaglia, Enrica; Fantoni, Luca; Grande, Alexis; Parenti, Sandra; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Tatiana Vignudelli, Tatiana; Montanari, Monica; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Bicciato, Silvio; Ferrari, Sergio
abstract
Acute Myeloid Leukemia (AML) blast cells are immature committed myeloid cells unable to spontaneously undergo terminal maturation, characterized by heterogeneous sensitivity to natural differentiation inducers. No data are available so far by which infer the AML’s response to differentiating therapy. Thus, we have initially profiled by GeneChip arrays the gene expression of several AML cell lines: they derived by the original blast cell populations and are still characterized by the same immunophenotype, retain a different sensitivity or resistance to All-Trans Retinoic-Acid (ATRA) and Vitamin-D3 (VD) and never undergo spontaneously terminal maturation. Here we show that differences exist by which predict the cell line differentiation fate. Next we constructed a signature able to predict resistance or sensitivity to the differentiation induction and tested it, using a TaqMan platform, for its capability to predict the in-vitro response of 28 VD or ATRA treated AML blast cell populations. Finally, by a meta-analysis of public available microarray data we demonstrated that our signature of 11 genes, among them is particularly intriguing the presence of Meis1 and ID3, that was formerly designed to identify differentiation therapy resistant populations, turned out to be a good classifier for clusters of patients known to have poor prognostic significance.
2006
- Identification of a molecular signature predictive of sensitivity to differentiation induction in acute myeloid leukemia
[Articolo su rivista]
Tagliafico, Enrico; Tenedini, Elena; Manfredini, Rossella; Grande, Alexis; Ferrari, F.; Roncaglia, Enrica; Bicciato, Silvio; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Gemelli, Claudia; Montanari, Monica; Vignudelli, Tatiana; ZANOCCO MARANI, Tommaso; Parenti, Sandra; Paolucci, Paolo; Martinelli, G.; Piccaluga, P. P.; Baccarani, M.; Specchia, G.; Torelli, U.; Ferrari, Sergio
abstract
Acute myeloid leukemia (AML) blasts are immature committed myeloid cells unable to spontaneously undergo terminal maturation, and characterized by heterogeneous sensitivity to natural differentiation inducers. Here, we show a molecular signature predicting the resistance or sensitivity of six myeloid cell lines to differentiation induced in vitro with retinoic acid or vitamin D. The identified signature was further validated by TaqMan assay for the prediction of response to an in vitro differentiation assay performed on 28 freshly isolated AML blast populations. The TaqMan assay successfully predicts the in vitro resistance or responsiveness of AML blasts to differentiation inducers. Furthermore, performing a meta-analysis of publicly available microarray data sets, we also show the accuracy of our prediction on known phenotypes and suggest that our signature could become useful for the identification of patients eligible for new therapeutic strategies.
2006
- Virally mediated MafB transduction induces the monocyte commitment of human CD34+ hematopoietic stem/progenitor cells
[Articolo su rivista]
Gemelli, Claudia; Montanari, Monica; Tenedini, Elena; ZANOCCO MARANI, Tommaso; Vignudelli, Tatiana; Siena, Michela; Zini, Roberta; Salati, Simona; Tagliafico, Enrico; Manfredini, Rossella; Specchia, G; Grande, Alexis; Ferrari, Sergio
abstract
Upregulation of specific transcription factors is a generally accepted mechanism to explain the commitment of hematopoietic stem cells along precise maturation lineages. Based on this premise, transduction of primary hematopoietic stem/progenitor cells with viral vectors containing the investigated transcription factors appears as a suitable experimental model to identify such regulators. Although MafB transcription factor is believed to play a role in the regulation of monocytic commitment, no demonstration is, to date, available supporting this function in normal human hematopoiesis. To address this issue, we retrovirally transduced cord blood CD34+ hematopoietic progenitors with a MafB cDNA. Immunophenotypic and morphological analysis of transduced cells demonstrated the induction of a remarkable monomacrophage differentiation. Microarray analysis confirmed these findings and disclosed the upregulation of macrophage-related transcription factors belonging to the AP-1, MAF, PPAR and MiT families. Altogether our data allow to conclude that MafB is a key regulator of human monocytopoiesis.
2005
- Correlation between differentiation plasticity and mRNA expression profiling of CD34+-derived CD14- and CD14+ human normal myeloid precursors
[Articolo su rivista]
Montanari, Monica; Gemelli, Claudia; Tenedini, Elena; ZANOCCO MARANI, Tommaso; Vignudelli, T; Siena, M; Zini, Roberta; Salati, Simona; Chiossi, G; Tagliafico, Enrico; Manfredini, Rossella; Grande, Alexis; Ferrari, Sergio
abstract
In spite of their apparently restricted differentiation potentiality, hematopoietic precursors are plastic cells able to trans-differentiate from a maturation lineage to another. To better characterize this differentiation plasticity, we purified CD14- and CD14+ myeloid precursors generated by 'in vitro' culture of human CD34+ hematopoietic progenitors. Morphological analysis of the investigated cell populations indicated that, as expected, they consisted of granulocyte and monocyte precursors, respectively. Treatment with differentiation inducers revealed that CD14- cells were bipotent granulo-monocyte precursors, while CD14+ cells appeared univocally committed to a terminal macrophage maturation. Flow cytometry analysis demonstrated that the conversion of granulocyte precursors to the mono-macrophage maturation lineage occurs through a differentiation transition in which the granulocyte-related myeloperoxidase enzyme and the monocyte-specific CD14 antigen are co-expressed. Expression profiling evidenced that the observed trans-differentiation process was accompanied by a remarkable upregulation of the monocyte-related MafB transcription factor.
2005
- IDENTIFICATION OF A MOLECULAR SIGNATURE PREDICTIVE OF REFRACTORINESS IN ACUTE MYELOID LEUKEMIA
[Abstract in Atti di Convegno]
Tagliafico, Enrico; Tenedini, Elena; Manfredini, Rossella; Ferrari, Sergio; Roncaglia, Enrica; Fantoni, Luca; Grande, Alexis; Parenti, Sandra; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Vignudelli, Tatiana; Montanari, Monica; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Bicciato, Silvio; Specchia, Giorgina; Martinelli, Giovanni; Baccarani, Michele; Piccaluga, Pier Paolo; Torelli, Umberto; Ferrari, Sergio
abstract
Acute Myeloid Leukemia (AML) blast cells are immature committed myeloid cells unable to spontaneously undergo terminal maturation, characterized by heterogeneous sensitivity to natural differentiation inducers. No data are available so far by which infer the AML’s response to differentiating therapy. Thus, we have initially profiled by GeneChip arrays the gene expression of several AML cell lines: they derived by the original blast cell populations and are still characterized by the same immunophenotype, retain a different sensitivity or resistance to ATRA and VD and never undergo spontaneously terminal maturation. Here we show that differences exist by which predict the cell line differentiation fate. Next we constructed a signature able to predict resistance or sensitivity to the differentiation induction and tested it, using a TaqMan platform, for its capability to predict the in-vitro response of 28 VD or ATRA treated AML blast cell populations. Finally, by a meta-analysis of public available microarray data we demonstrated that our signature, that was formerly designed to identify differentiation therapy resistant populations, turned out to be a good classifier for clusters of patients with citogenetically and molecularly defined lesions that are known to have poor prognostic significance.
2005
- The kinetic status of hematopoietic stem cell subpopulations underlies a differential expression of genes involved in self-renewal, commitment, and engraftment.
[Articolo su rivista]
Manfredini, Rossella; Zini, Roberta; Salati, Simona; Siena, M; Tenedini, Elena; Tagliafico, Enrico; Montanari, Monica; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Vignudelli, T; Grande, A; Fogli, M; Rossi, L; Fagioli, Me; Catani, L; Lemoli, Rm; Ferrari, Sergio
abstract
The gene expression profile of CD34(-) hematopoietic stem cells (HSCs) and the correlations with their biological properties are still poorly understood. To address this issue, we used the DNA microarray technology to compare the expression profiles of different peripheral blood hemopoietic stem/progenitor cell subsets, lineage-negative (Lin(-)) CD34(-), Lin(-)CD34(+), and Lin(+)CD34(+) cells. The analysis of gene categories differentially expressed shows that the expression of CD34 is associated with cell cycle entry and metabolic activation, such as DNA, RNA, and protein synthesis. Moreover, the significant upregulation in CD34(-) cells of pathways inhibiting HSC proliferation induces a strong differential expression of cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and growth-arrest genes. According to the expression of their receptors and transducers, interleukin (IL)-10 and IL-17 showed an inhibitory effect on the clonogenic activity of CD34(-) cells. Conversely, CD34(+) cells were sensitive to the mitogenic stimulus of thrombopoietin. Furthermore, CD34(-) cells express preferentially genes related to neural, epithelial, and muscle differentiation. The analysis of transcription factor expression shows that the CD34 induction results in the upregulation of genes related to self-renewal and lineage commitment. The preferential expression in CD34(+) cells of genes supporting the HSC mobilization and homing to the bone marrow, such as chemokine receptors and integrins, gives the molecular basis for the higher engraftment capacity of CD34(+) cells. Thus, the different kinetic status of CD34(-) and CD34(+) cells, detailed by molecular and functional analysis, significantly influences their biological behavior
2003
- Development of an IL-6 antagonist peptide that induces apoptosis in IL-6 dependent 7TD1 cells.
[Articolo su rivista]
Manfredini, Rossella; Tenedini, Elena; M., Siena; Tagliafico, Enrico; Montanari, Monica; Grande, Alexis; ZANOCCO MARANI, Tommaso; C., Poligani; Zini, Roberta; A., Bergamaschi; DE RIENZO, Francesca; DE BENEDETTI, Pier Giuseppe; Menziani, Maria Cristina; Ferrari, Sergio
abstract
Interleukin-6 (IL-6) is a pleiotropic cytokine involved in the regulation of proliferation and differentiation of hematopoietic cells and in the pathogenesis of many diseases, including multiple myeloma. This study pursues a way to interfere with IL-6 pathway in an attempt to modulate its biological activity. Here we describe the rational design and biological evaluation of peptides able to antagonize the murine IL-6 activity by interfering with IL-6 Receptor alpha in 7TD1 cells, a IL-6-dependent B-cell line. Of the peptide tested, only Guess 4a is capable of interfering with IL-6 transducing pathway, therefore inducing growth arrest and apoptosis of 7TD1 cells.
2003
- Requirement of the coiled-coil domains of p92(c-Fes) for nuclear localization in myeloid cells upon induction of differentiation
[Articolo su rivista]
Tagliafico, Enrico; M., Siena; ZANOCCO MARANI, Tommaso; Manfredini, Rossella; Tenedini, Elena; M., Montanari; Grande, Alexis; Ferrari, Sergio
abstract
The nonreceptor tyrosine kinase Fes is implicated in myeloid cells differentiation. It has been observed that its localization can be cytoplasmic, perinuclear, or nuclear. To further characterize this point, we studied Fes subcellular localization in myeloid cell lines (HL60 and K562) and in COS1 cells. Fes was observed in both the nucleus and the cytoplasm of HL60, K562 cells over-expressing Fes and only in the cytoplasm of COS1 cells, suggesting that nuclear localization is cell context dependent. Moreover, in myeloid cells, the treatment with differentiation-inducing agents such as retinoic acid, phorbol esters and vitamin D, is followed by an increase of the oligomeric form of Fes in the nucleus. In fact, oligomerization seems to be necessary for translocation to occur, since Fes mutants missing the coiled-coil domains are not able to form oligomers and fail to localize in the nucleus. The active form of Fes is tyrosine phosphorylated; however, phosphorylation is not required for Fes to localize in the nucleus, since tyrosine kinase inhibitors do not block the translocation process.
2002
- Gene Expression profile of Vitamin D3 treated HL60 cells shows a phenotypic but not a complete functional conversion to monocytes
[Abstract in Atti di Convegno]
Tenedini, Elena; Bergamaschi, A; Manfredini, Rossella; Percudani, R; Siena, M; ZANOCCO MARANI, Tommaso; Grande, Alexis; Montanari, Monica; Gemelli, Claudia; Torelli, U; Ferrari, Sergio; Tagliafico, Enrico
abstract
Acute Myeloid leukemia blast cells are characterized by their inability to proceed spontaneously toward terminal differentiation. To tackle this problem we have studied the changes occurring in the gene expression profile during the differentiation of HL60 cells treated with VD using the Affymetrix GeneChip technology and we have compared the molecular phenotype of VD induced cells to that of CD14+ pheripheral monocytes.
2002
- Gene expression profile of vitamin D3 treated HL60 cells shows an incomplete molecular phenotypic conversion to monocytes
[Articolo su rivista]
Tagliafico, Enrico; Tenedini, Elena; A., Bergamaschi; ZANOCCO MARANI, Tommaso; R., Percudani; M., Siena; Manfredini, Rossella; Grande, Alexis; M., Montanari; C., Gemelli; U., Torelli; Ferrari, Sergio
abstract
By high density oligonucleotide microarrays we have studied the expression profile of proliferating and VD treated HL60 cells and the molecular phenotype of VD monocytes and that of CD14+ peripheral monocytes has been compared. The results indicate that important changes in functional categories of the differentially expressed genes underlie the differentiation transition from myeloblasts to monocytes. This differential gene expression pattern leads to an increased expression of mRNAs involved in surface and external activities since many of the VD induced genes belong to ligand binding, receptors, cell surface antigens, defense/immunity and adhesion molecules functional categories. results also indicate that the molecular phenotypes of monocytes and VD induced cells diverge for a small but significant set of defense related genes. Particularly, class II MHC genes are not expressed in these cells. Furthermore, the high levels of expression of these genes induced by serum treatment of monocytes are decreased by VD.
2002
- Physiological levels of 1 alpha, 25 di-hydroxyvitamin D3 induce the monocytic commitment of CD34+ hematopoietic progenitors
[Articolo su rivista]
Grande, Alexis; M., Montanari; Tagliafico, Enrico; Manfredini, Rossella; ZANOCCO MARANI, Tommaso; M., Siena; Tenedini, Elena; A., Gallinelli; Ferrari, Sergio
abstract
Although supraphysiological levels of 1alpha, 25 dihydroxyvitamin D3 (VD) have been demonstrated extensively to induce the monomacrophagic differentiation of leukemic myelo- and monoblasts, little is known about the role that physiological levels of this vitamin could play in the regulation of normal hematopoiesis. To clarify this issue, we adopted a liquid-culture model in which cord blood CD34+ hematopoietic progenitors, induced to differentiate in the presence of different combinations of cytokines, were exposed to VD at various concentrations and stimulation modalities. The data obtained show that physiological levels of VD promote a differentiation of CD34+ hematopoietic progenitors characterized by the induction of all the monomacrophagic immunophenotypic and morphological markers. This effect is not only exerted at the terminal maturation but also at the commitment level, as demonstrated by the decrease of highly undifferentiated CD34+CD38-hematopoietic stem cells, the down-regulation of CD34 antigen, and the increase of monocyte-committed progenitors. Molecular analysis suggests that the VD genomic signaling pathway underlies the described differentiation effects.
2002
- Requirement of the coiled coil domains of p92c-Fes for nuclear translocation in myeloid cells upon induction of differentiation
[Abstract in Atti di Convegno]
ZANOCCO MARANI, Tommaso; Siena, M; Tagliafico, Enrico; Manfredini, Rossella; Tenedini, Elena; Montanari, Monica; Grande, Alexis; Gemelli, Claudia; Ferrari, Sergio
abstract
The non-receptor tyrosine kinase Fes is expressed in hematopoietic progenitors, differentiated myeloid cells and other cell types, such as vascular endothelial cells and neuroblastoma cell lines. To further clarify this point we performed confocal microscopy and western blot experiments on myeloid cell lines and COS1 cells. In myeloid cells the treatment with differentiation inducing agents such as ATRA, PMA and VD is followed by an increase of Fes abundance in the nuclear compartment. The active form of Fes is phosphorylated on residue 713 and is present into the nucleus while treated cells with the tyrosine kinase inhibitor Genistein clearly showed that phosphorylation is not a required event in order to Fes to translocate to the nucleus.
2001
- A functionally active RAR alpha nuclear receptor is expressed in retinoic acid non responsive early myeloblastic cell lines
[Articolo su rivista]
Grande, Alexis; M., Montanari; Manfredini, Rossella; Tagliafico, Enrico; ZANOCCO MARANI, Tommaso; F., Trevisan; Ligabue, Giulia; M., Siena; Ferrari, Stefano; Ferrari, Sergio
abstract
Although ail-trans retinoic acid (ATRA) can restore the differentiation capacity of leukemic promyelocytes, early leukemic myeloblasts are conversely not responsive to ATRA induced granulocytic differentiation. To assess whether this resistance to ATRA is related to an impaired function of the Retinoic Acid Receptor alpha (RAR alpha), we performed an analysis of RAR alpha expression and transactivation activity, in several myeloid leukemic cell lines, representative of different types of spontaneous acute myeloid leukemias. Our results indicate that a functionally active RAR alpha nuclear receptor is expressed in all the analyzed cell lines, regardless of their differentiation capacity following exposure to ATRA. The observation that ATRA treatment is able to induce the expression of retinoic acid target genes, in late- but not in early-myetoblastic leukemic cells, raises the possibility that the differentiation block of these cells is achieved through a chromatin mediated mechanism. Acetylation is apparently not involved in this process, since the histone deacetylase inhibitor trichostatin A, is not able to restore the differentiation capacity of early leukemic myeloblasts. Further investigation is needed to clarify whether myeloid transcription factors, distinct to RAR alpha, play a role in the resistance of these cells to ATRA treatment.
1999
- Induction of a functional vitamin D receptor in all-trans-retinoic acid- induced monocytic differentiation of M2-type leukemic blast cells
[Articolo su rivista]
Manfredini, R.; Trevisan, F.; Grande, A.; Tagliafico, E.; Montanari, M.; Lemoli, R.; Visani, G.; Tura, S.; Ferrari, S.; Ferrari, S.
abstract
Different types of acute myeloid leukemia blast cells were induced to differentiate in vitro with all-trans-retinoic acid (ATRA) and vitamin D3 (VD). M0/M1 leukemic cells are not sensitive to differentiating agents, whereas M3 leukemic cells are induced to undergo granulocytic differentiation after ATRA treatment but are not sensitive to VD. M2 leukemic blast cells behave differently because they undergo monocytic differentiation with both the differentiation inducers. To gain some insight into the maturation of M2- type leukemic cells, we studied the molecular mechanisms underlying monocytic differentiation induced by ATRA and VD in spontaneous M2 blast cells as well as in Kasumi-1 cells (an acute myeloid leukemia M2-type cell line). Our results indicate that ATRA as well as VD efficiently increases the nuclear abundance of VD receptor (VDR) and promotes monocytic differentiation. VDR is functionally active in ATRA-treated Kasumi-1 cells because it efficiently heterodimerizes with retinoid X receptor, binds to a DR3-type vitamin D- responsive element, and activates the transcription of a vitamin D-responsive element-regulated reporter gene. Consistent with these findings, VD- responsive genes are induced by ATRA treatment of Kasumi-1 cells, suggesting that the genetic program underlying monocytic differentiation is activated. The molecular mechanism by which ATRA increases the nuclear abundance of a functional VDR is still unknown, but our data clearly indicate that the M2 leukemic cell context is only permissive of monocytic differentiation.
1998
- Antisense inhibition of Bax mRNA increases survival of terminally differentiated HL60 cells
[Articolo su rivista]
Manfredini, R.; Capobianco, M. L.; Trevisan, F.; Rauzi, F.; Barbieri, D.; Citro, G.; Tagliafico, E.; Ferrari, S.
abstract
Cell sensitivity to programmed cell death is primarily modulated by members of the Bcl-2 family, as the balance of homodimer or heterodimer formation between proapoptotic and antiapoptotic members defines apoptosis susceptibility in the great majority of cellular contexts. It is, therefore, important to clarify if the Bax protein is limiting for activation of the genetic program of programmed cell death or can be complemented by different Bcl-2 family members, such as Bah or Bad. To gain some insight into the role of Bax in the molecular mechanisms of apoptosis of myeloid cells, we inhibited this gene in all-trans-retinoic acid (ATRA)-treated HL60 cells using the methodology of antisense oligodeoxynucleotides (AS-ODN). Our results indicate that Bax inhibition has no effect on the proliferation and differentiation capacity of HL60 cells. Instead, the survival rate of terminally differentiated Bax-inactivated HL60 (Bax((-)) HL60) cells is almost three times higher in respect to control cultures, indicating that in mature granulocytes Bax is not efficiently complemented by others members of the Bcl-2 family proteins.
1997
- Antisense inhibition of c-fes proto-oncogene blocks PMA-induced macrophage differentiation in HL60 and in FDC-P1/MAC-11 cells
[Articolo su rivista]
Manfredini, Rossella; R., Balestri; Tagliafico, Enrico; F., Trevisan; Grande, Alexis; M., Pizzanelli; D., Barbieri; P., Zucchini; G., Citro; C., Franceschi; Ferrari, Sergio
abstract
To gain some insight into the role of c-fes in macrophage differentiation, we have analyzed the ability of HL60 leukemic promyelocytic cells and FDC-P1/MAC-11 murine myeloid precursor cells to differentiate in response to phorbol esters after inhibition of c-fes function. Fes inactivation has been obtained by using oligodeoxynucleotides (ODN) complementary to the 5´ region of c-fes mRNA and to 5´ splice junctions of c-fes primary transcript. After 5 days (d) in culture, in several separate experiments performed with different ODN preparations, a complete inhibition of c-fes expression was observed in HL60 and in FDC-P1/MAC-11 cells. No perturbation of cell growth was evident in our experimental conditions in both cell lines after c-fes inhibition. Furthermore, in HL60 cells lacking c-fes product, an almost complete downregulation of the alpha 4 beta 1 fibronectin receptor occurred. However, in both cell lines, the induction of macrophage differentiation by phorbol esters resulted in an almost complete maturation arrest as evaluated by morphological, cytochemical, immunological criteria, and by the cytofluorimetric cell cycle analysis. A loss of the adhesion capacity of both myeloid cell lines, when compared to terminally differentated macrophages, was also observed. These results suggest that HL60 and FDC-P1/MAC-11 cells, when treated with phorbol 12-myristate 13-acetate, require c-fes protein expression to activate the genetic program underlying macrophage differentiation.
1997
- Correlations between the FAB phenotype and differentiation potential in leukemic myelopoiesis
[Relazione in Atti di Convegno]
Manfredini, R.; Trevisan, F.; Grande, A.; Tagliafico, E.; Montanari, M.; Pignatti, E.; Ligabue, G.; Barbieri, D.; Ferrari, Se.
abstract
1997
- Presence of a functional vitamin D receptor does not correlate with vitamin D3 phenotypic effects in myeloid differentiation
[Articolo su rivista]
Grande, Alexis; Manfredini, Rossella; M., Pizzanelli; Tagliafico, Enrico; R., Balestri; F., Trevisan; D., Barbieri; C., Franceschi; Battini, Renata; Ferrari, Stefano; Ferrari, Sergio
abstract
Although VDR is expressed in all the acute myeloid leukemia cell populations studied, most of these leukemias do not exibit any phenotypic response when exposed to VD. To determine whether VD resistance is related to an altered VDR function,we performed an analysis of VDR expression, phosphorylation, DNA binding capacity and transactivation activity in several leukemic myeloid cell lines arrested at different levels of maturation. Our results indicate that VD induces a clear phenotypic effect, i.e. terminal monocytic differentiation, only in leukemic cells of M2/M3 (intermediate myeloblasts) and M5 (monoblasts) types but not in erythroid precursor cells, early leukemic myeloblasts (M0/M1 type) and promyelocytes (M3 type). VDR expression and function are evident in all the nuclear extracts obtained from the different myeloid cell lines after 12 h of VD treatment, but VD activation of monocytic differentiation is limited to a narrow differentiation window characterized by the M2 type myeloid cellular context.
1996
- Inhibition of cell growth by accumulated spermine is associated with a transient alteration of cell cycle progression
[Articolo su rivista]
Monti, M. G.; Pernecco, L.; Manfredini, R.; Frassineti, C.; Barbieri, D.; Marverti, G.; Ghiaroni, S.
abstract
Exposure of HL-60 cells to millimolar levels of spermine resulted in the inhibition of cell growth. Flow cytometry revealed that the addition of exogenous spermine prevented the accumulation of cells in the S and G2/M phases of the cell cycle as observed in the control cells. High intracellular levels of spermine completely suppressed the early onset of ornithine decarboxylase activity and, consequently, the intracellular increase in spermidine and putrescine. On the other hand, the addition of exogenous spermidine or putrescine also abolished ornithine decarboxylase activity, but in this case neither the growth of spermidine or putrescine-treated cells nor the cell cycle phase distribution was affected. In the latter cells, intracellular levels of spermidine were not significantly different from control ones. These results suggest that the addition of exogenous spermine inhibits cell proliferation by hindering the increase in cellular spermidine needed to accelerate the G, to S phase transition.
1995
- All-trans-retinoic acid induces simultaneously granulocytic differentiation and expression of inflammatory cytokines in HL-60 cells
[Articolo su rivista]
Grande, Alexis; Manfredini, Rossella; Tagliafico, Enrico; R., Balestri; M., Pizzanelli; S., Papa; P., Zucchini; L., Bonsi; G., Bagnara; U., Torelli; Ferrari, Sergio
abstract
All-trans-retinoic acid (ATRA) can induce granulocytic differentiation both in vitro and in vivo, and its activity is mediated by the retinoic acid receptor-alpha (RAR-alpha). In the present study, we evaluated the ability of this inducer in HL-60 cells, to stimulate simultaneously granulocytic differentiation and the expression of the cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-3, IL-6, tumor necrosis factor-a: (TNF-a), and stem cell factor (SCF). The level of expression of these cytokines in ATRA-treated HL-60 cells was compared with that observed in normal and lipopolysaccharide (LPS)-heated peripheral granulocytes. The results indicate that the expression of these cytokines is enhanced during differentiation so that the pattern observed in ATRA-treated HL-60 cells is close to that of LPS-stimulated normal granulocytes. In addition, tetra phorbol acetate (TPA)-treated HL-60 cells express several of the above listed cytokines. It is concluded that ATRA not only induces granulocytic differentiation of HL-60 cells, but also activation of these terminally differentiated cells. The activating cytokine expression in these cells appears related to the progress of the differentiation program induced by ATRA since normal granulocytes do not respond to this inducer by activation of the expression of these genes. Furthermore, the cytokine activation is a specific effect of ATRA, since DMSO does not have any stimulatory effect.
1995
- ROLE OF C-FES PROTOONCOGENE IN MYELOID DIFFERENTIATION
[Articolo su rivista]
FERRARI, Sergio; GRANDE, Alexis; MANFREDINI, Rossella; TAGLIAFICO, Enrico
abstract
The main purpose of this report is to provide a review of the present knowledge on the structure, function, and possible regulatory role of c-fes in the genetic programs underlying the proliferation and differentiation of hematopoietic myeloid cells. Fes encodes a non-receptor tyrosine kinase that is highly expressed in immature and differentiated cells of the granulocytic and mono-macrophagic lineages. It is therefore possible that c-fes is involved in the signal transduction of myeloid cell differentiation, even if the specific substrates phosphorylated by this protooncogene are only poorly characterised. Several experimental models have been established to evaluate the role of c-fes in myeloid differentiation, in particular: the differentiation capacity of HL60 cells lacking the p92(c-fes) protein, the transfection of c-fes gene into K562 cells and transgenic animals overexpressing c-fes, The results obtained point to the importance of c-fes in myeloid cells, since it appears to be involved in granulocytic maturation as an antiapoptotic gene, and in macrophagic maturation as a regulatory gene.
1994
- ANTISENSE STRATEGIES IN LEUKEMIA
[Articolo su rivista]
Ferrari, Sergio; Manfredini, Rossella; Torelli, U.
abstract
1994
- Antiapoptotic effect of c-fes protooncogene during granulocytic differentiation
[Articolo su rivista]
Ferrari, Sergio; Manfredini, Rossella; Tagliafico, Enrico; Grande, Alexis; D., Barbieri; R., Balestri; M., Pizzanelli; Zucchini, Patrizia; G., Citro; G., Zupi
abstract
The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation. Its product, p92c-fes, exhibits a tyrosine-kinase activity and is involved in the cellular response to GM-CSF, but its role is not yet clarified. To study this problem, the c-fes protooncogene expression has been inhibited in HL60 cells and in fresh leukemic blast cells of Acute Promyelocytic Leukemia (APL) induced to differentiate with All-Trans-Retinoic Acid (ATRA). Inhibition of c-fes function was obtained by treatment of the cells with a specific antisense oligomer complementary to the 5' region of the c-fes mRNA. It was observed that the cells, rather then differentiate to granulocytes, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. Superimposable results are obtained on blast cells from APL. It is possible to conclude that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of c-fes expression and treatment with ATRA, is due to activation of programmed cell death rather than an accelerated differentiation. Our data suggest that the c-fes product is essential for the survival of myeloid cells during differentiation.
1994
- Cultured Normal Human Keratinocytes Secrete Stem-Cell Factor And Express C-Kit Receptor
[Abstract in Rivista]
G., Zambruno; Marconi, Alessandra; Manfredini, Rossella; D., Ottani; M., Pizzanelli; Giannetti, Alberto; G., Girolomoni
abstract
nd
1994
- Expression And Function Of Nerve Growth-Factor And Nerve Growth-Factor Receptor On Cultured Keratinocytes.
[Articolo su rivista]
Pincelli, Carlo; C., Sevignani; Manfredini, Rossella; Grande, Alexis; F., Fantini; L., Bracci Laudiero; L., Aloe; Ferrari, Sergio; Cossarizza, Andrea; Giannetti, Alberto
abstract
Keratinocytes, a key cellular component both for homeostasis and pathophysiologic processes of the skin, secrete a number of cytokines and are stimulated by several growth factors. Nerve growth factor (NGF) is synthesized in the skin and basal keratinocytes express the low-affinity nerve growth factor receptor (NGF-R). We present evidence that normal human keratinocytes in culture express the low- and the high-affinity NGF-R both at the mRNA level, as determined by reverse-transcription polymerase chain reaction and at the protein level, as shown by cytofluorimetric analysis. NGF significantly stimulates the proliferation of normal human keratinocytes in culture in a dose-dependent manner. This effect can be prevented by the addition of both an anti-NGF neutralizing antibody and a high-affinity NGF-R (trk) specific inhibitor, the natural alkaloid K252a. By contrast, keratinocyte proliferation is not inhibited by an anti - low-affinity NGF-R monoclonal antibody, thus suggesting that NGF effect on human keratinocytes is mediated by the high-affinity NGF-R. Moreover, NGF mRNA is expressed in normal human keratinocytes and NGF is secreted by keratinocytes in increasing amounts during growth, as detected by enzyme-linked immunosorbent assay. These results suggest that NGF could act as a cytokine in human skin and take part in disorders of keratinocyte proliferation.
1994
- Expression of B7 costimulatory molecule in cultured human epidermal Langerhans cells is regulated at the mRNA level.
[Articolo su rivista]
Girolomoni, G; Zambruno, G; Manfredini, Rossella; Zacchi, V; Ferrari, Sergio; Cossarizza, Andrea; Giannetti, A.
abstract
Langerhans cells (LC) belong to the dendritic cell lineage and are the principal antigen-presenting cells of squamous epithelia. Short-term cultured LC (cLC) exhibit a marked augmented capacity to stimulate allogeneic T cells and acquire the ability to activate naive T cells, probably in relation to enhanced expression of accessory signals. In this study, we evaluated the expression of B7 costimulatory molecule (CD80) in human freshly isolated (fLC) and cLC at both the protein and mRNA level. Staining of frozen skin sections did not reveal any epidermal dendritic cell reactive with either of two different anti-B7 monoclonal antibodies. fLC in suspension did not exhibit any B7 staining as evaluated by two-color flow-cytometry analysis and immunoelectron microscopy. In contrast, LC that were cultured for 24-72 h displayed strong surface B7 reactivity with a characteristic patchy pattern. Treatment with dispase and trypsin did not reduce B7 staining of cLC. Following warming to 37°C, cLC tagged with anti-B7 monoclonal antibody and gold-conjugated secondary antibody could internalize surface B7 by using the organelles of receptor-mediated endocytosis. B7 mRNA, detected by the reverse-transcriptase polymerase chain reaction technique, was expressed at a low level in purified (> 90% HLA-DR+) fLC but not in LC-depleted epidermal cells, and was markedly upregulated in purified cLC. The results indicate that 1) fLC do not express B7 protein on their surface, but acquire B7 during culture, 2) surface B7 is not sensitive to trypsin, 3) B7 expression is regulated primarily at the mRNA level, and 4) membrane B7 can be internalized within cLC. B7 molecule on CLC may be relevant to their increased antigen-presenting cell potency and ability to stimulate naive T lymphocytes.
1993
- Antisense strategies in leukemias
[Articolo su rivista]
S., Ferrari; Manfredini, Rossella; U., Torelli
abstract
The experimentation of antisense oligos isrecently been carried out in laboratory animalsand in clinical trials of patients having differenthematological neoplastic diseases. In the futureit will be probably possible to reach one of themore desired goal in cancer therapy: specificityof act ion and s e l e c t ive ce l l d e a th o f c ance rcells.
1993
- Expression of interleukins 1, 3, 6, stem cell factor and their receptors in acute leukemia blast cells and in normal peripheral lymphocytes and monocytes
[Articolo su rivista]
Ferrari, S.; Grande, A.; Manfredini, R.; Tagliafico, E.; Zucchini, P.; Torelli, G.; Torelli, U.
abstract
Abstract: Reverse transcriptase‐polymerase chain reaction amplification (RT‐PCR) and Southern blot analysis were used to evaluate ligand and receptor expression of interleukin 1α (IL‐1α), interleukin 3 (IL‐3), interleukin 6 (IL‐6) and stem cell factor (SCF) in peripheral blood lymphocytes and monocytes and in several acute leukemia blast cell populations. Resting peripheral lymphocytes and monocytes expressed both ligand and receptor of the four cytokines at considerable levels. The leukemic blast cells of the M1‐M4 phenotypes are characterized by almost complete lack of expression of IL‐1α, IL‐3 and IL‐6 and the constant and usually high expression of SCF. On the other hand, these myeloid blast cells express generally high levels of the four cytokine receptors. The data suggest that the regulation of the expression of IL‐1α, IL‐3 and IL‐6, at least in our limited number of leukemic cell populations studied, is independent of that of SCF. The results indicate that, at least in most of the leukemic myeloid blasts cells, the expression of SCF and its receptor, the c‐kit oncogene, may permit an autocrine regulation of cell cycling. © Munksgaard 1993
1993
- Inhibition of c-fes expression by an antisense oligomer causes apoptosis oh HL60 cells induced to granulocytic differentiation
[Articolo su rivista]
Manfredini, Rossella; Grande, Alexis; Tagliafico, Enrico; D., Barbieri; P., Zucchini; G., Citro; G., Zupi; C., Franceschi; U., Torelli; Ferrari, Sergio
abstract
The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation, but so far no definite function has been attributed to the product of this oncogene. To tackle this problem, the c-fes protooncogene expression has been inhibited in HL60 cells, and fresh leukemic promyelocytes of acute promyelocytic leukemia have been induced to differentiate with retinoic acid (RA) and dimethylsulfoxide (DMSO). Inhibition was obtained by incubating the cells with a specific c-fes antisense oligodeoxynucleotide. It was observed that the cells, rather than differentiating, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. This process was inhibited by granulocyte and granulocyte/macrophage colony-stimulating factor, but not by interleukin 3 (IL-3), IL-6, or stem cell factor. Our present results demonstrate that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of the c-fes gene product and treatment with RA-DMSO, is due to activation of programmed cell death. It is concluded that a possible role of the c-fes gene product is to exert an antiapoptotic effect during granulocytic differentiation.
1993
- Overexpression of c-kit in a leukemic cell population carrying a trisomy 4 and its relationship with the proliferative capacity
[Articolo su rivista]
Ferrari, S.; Grande, A.; Zucchini, P.; Manfredini, R.; Tagliafico, E.; Rossi, E.; Temperani, P.; Torelli, G.; Emilia, G.; Torelli, U.
abstract
The expression of c-kit and its ligand, the stem cell factor (SCF), was studied in five cases of acute myeloid leukemia. One of these had a trisomy of chromosome 4, where the c-kit oncogene is located. In this case, the c-kit oncogene was overexpressed, but matched by a low expression of its ligand, SCF. The molecular evaluation of the growth rate by c-myc and the histone H3 expression indicated that the growth fraction of this cell population was very low. In one of the other leukemic cell populations studied, characterized by a low expression of c-kit and an elevated expression of the SCF, the growth fraction was also very low. Our results suggest that at least for some receptor oncogenes, the simple overexpression cannot be taken as an indication that the oncogene is involved in the deregulation of cell proliferation. © 1993 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
1992
- ANTISENSE STRATEGIES TO CHARACTERIZE THE ROLE OF GENES AND ONCOGENES INVOLVED IN MYELOID DIFFERENTIATION
[Articolo su rivista]
Ferrari, Sergio; Manfredini, Rossella; Grande, Alexis; Torelli, Umberto
abstract
Several genes that ara associated with cell proliferation and differentiation have been isolated. The definition of "cell cycle-related or differentiation-related genes" is simply based on the concept that the mRNA and/or protein abundance of these genes increases in these two functional states of the cell .....
1992
- Abundance of the Primary Transcript and Its Processed Product of Growth-related Genes in Normal and Leukemic Cells during Proliferation and Differentiation
[Articolo su rivista]
Ferrari, S.; Tagliafico, E.; Manfredini, R.; Grande, A.; Rossi, E.; Zucchini, P.; Torelli, G.; Torelli, U.
abstract
The relative abundance of primary transcript and mature mRNA of the c-myc, calcyclin, S14 ribosomal protein, and rRNA genes was deter-mined densitometrically after reverse transcriptase-polymerase chain reaction and Northern blotting analysis in resting and mitogen-stimulated lymphocytes, proliferating and terminally differentiated HL-60 cells, and leukemic blast cells. Transcription and processing of c-myc and rRNA gene transcripts increased proportionally after mitogen stimulation, whereas these processes were independent of cell cycling status in the case of the SI 4 gene. Normal lymphocytes showed an unexpectedly large amount of primary transcript of the calcyclin gene, whereas the corresponding mRNA was undetectable. The abundance of c-myc, calcyclin, and S14 mRNA in terminally differentiated HL-60 cells decreased sharply, compared to their proliferating counterparts. This decrease reflected post-transcriptional modulation, since the abundance of precursor remained essentially unchanged. After HL-60 differentiation, the 32S rRNA levels remained relatively high, whereas the 45S primary transcript almost disappeared. Leukemic blast cells displayed highly variable precursor/mRNA ratios of c-myc, calcyclin, and S14 genes but consistently high ratios of 32S to 45S RNA, suggesting that the cleavage rate of the 32S rRNA was sharply reduced in these cells, resulting in an accumulation of this molecule. These results suggest the importance of efficient processing of primary transcript to generate adequate functional mRNA, thus regulating gene expression. Furthermore, in terminally differentiated cells and leukemic blast cells a stabilization of the primary transcript without efficient processing can be observed. The role of the stabilization of the primary transcript in terminal differentiation is further supported by the results of run-off transcription, indicating a sharp decrease of c-myc and calcyclin transcription rate in retinoic acid/dimethyl sulfoxide-treated HL-60 cells. © 1992, American Association for Cancer Research. All rights reserved.
1992
- Proliferation, Differentiation Arrest, and Survival in Leukemic Blast Cells
[Articolo su rivista]
Ferrari, S.; Manfredini, R.; Grande, A.; Torelli, G.; Torelli, U.
abstract
1992
- Terminal differentiation
[Articolo su rivista]
Ferrari, Sergio; Grande, Alexis; Manfredini, Rossella; Torelli, Umberto
abstract
Cell differentiation ultimately proceeds as a response to signals from the extracellu- lar microenvironment. If the cells are competent, that is, if they carry the correspond- ing receptors and signal transduction pathways, they can be triggered in the commit- ment state. In many cases the response includes complex gene expression programs whose progress and completion are relatively autonomous and whose outcome is determined by inherent regulatory factors of the cell type affected.' As a short introduction to the problems of differentiation and aging, we survey the main points that characterize the differentiation of hematopoietic cells. We can identify several main aspects.
1990
- Detection of low abundance mRNA of myeloid specific genes in cells of acute and chronic lymphoid leukemias by cRNA hybridization
[Articolo su rivista]
Ferrari, S.; Ceccherelli, G.; Tagliafico, E.; Zucchini, P.; Manfredini, R.; Torelli, G.; Emilia, G.; Torelli, U.
abstract
The hybridization to a complementary RNA (cRNA) probe both in situ and in solution was used to assay tiny amounts of mRNA of the lactoferrin (LF) and myeloperoxidase (MPO) genes in normal bone marrow cells and in acute and chronic lymphoid leukemias. Evidence is reported that this technique is much more sensitive than the standard Northern blot technique. The LF mRNA was detectable in three of seven cases of acute lymphoblastic leukemia (ALL) and in three of seven cases of chronic lymphocytic leukemia (CLL). Four cases of ALL were also positive when tested with the MPO cRNA. It is apparent from these results that myeloid specific mRNA, different from MPO, may be detected in leukemic cells with lymphoid phenotype using a method more sensitive than the Northern blot technique. Whether or not the molecular events observed in these cell populations reflect events physiologically occurring rather than a deregulation of gene expression associated to leukemogenesis remains to be established.
1990
- Differential effects of c-myb and c-fes antisense oligodeoxynucleotides on granulocytic differentiation of human myeloid leukemia HL60 cells
[Articolo su rivista]
Ferrari, S.; Donelli, A.; Manfredini, R.; Sarti, M.; Roncaglia, R.; Tagliafico, E.; Rossi, E.; Torelli, G.; Torelli, U.
abstract
To gain some insight into the role of c-myb and c-fes in myeloid differentiation, the authors have analyzed the ability of HL60 cells to differentiate in response to several different inducers after inhibition of c-myb and c-fes function. This function has been inhibited almost completely by using deoxynucleotides complementary to two 18-nucleotide sequences of c-myb and c-fes encoding mRNA. After 5 days in culture, in several separate experiments with different oligomer preparations, more than 90% growth inhibition was observed in c-myb antisense-treated HL60 cells. At this time, independent of the differentiation inducer used, c-myb antisense-treated HL60 cells differentiate only along the monocytic pathway, whereas in sense oligomer-treated cultures, retinoic acid and dimethyl sulfoxide induced granulocytic differentiation. No perturbation of the HL60 cell growth was observed after 5 days of treatment with antisense c-fes oligomer. However, induction to granulocytic differentiation by retinoic acid and dimethyl sulfoxide resulted in progressive cell death, whereas monocytic differentiation by other differentiation inducers was only marginally affected. These results suggest that granulocytic, unlike monocytic, differentiation requires c-myb-conditioned proliferation and the activity of the protein encoded by c-fes.
1990
- MITOCHONDRIAL-DNA DELETION IN A PATIENT WITH PROGRESSIVE OPHTHALMOPLEGIA
[Articolo su rivista]
Luppi, Mario; Marasca, Roberto; Sola, P; Corradi, M; Fancinelli, M; Montorsi, M; Manfredini, Rossella; Selleri, L.
abstract
Mitochondria are unique among intracellular organelles because they contain their own DNA, which can be transcribed and translated to form proteins. Mitochondrial diseases include myopathies and multisystem disorders. The case of a patient showing bilateral ophthalmoplegia with proximal limb weakness, severe dysphagia and short stature, without family history, is described. The analysis of mitochondrial DNA of the patient muscle revealed a deleted form accounting for 65% of the total mitochondrial DNA. The Southern Blot Analysis of mtDNA allows a rather precise localization of deletions giving new insights in the pathogenesis of mitochondrial myopathies and representing a new precious diagnostic tool in these diseases.
1990
- Noncoordinated Expression of S6, S11, and S14 Ribosomal Protein Genes in Leukemic Blast Cells
[Articolo su rivista]
Ferrari, S.; Manfredini, R.; Tagliafico, E.; Rossi, E.; Donelli, A.; Torelli, G.; Torelli, U.
abstract
The steady state levels of mRNAs codying for the ribosomal proteins S6, Sil, and SI4 have been evaluated in quiescent and proliferating human fibroblasts and in resting and proliferating human peripheral blood mononuclear cells. It was found that the amounts of ribosomal protein mRN A are very similar and are not increased by serum or mitogen stimulation. The constitutive expression of these genes appears to be coordinately regulated and it is not modified after protein synthesis inhibition by cycloheximide. The ribosomal protein mRNA was also assayed in 15 different populations of human leukemic blast cells. In these populations the abundance of each ribosomal protein mRNA is remarkably different from the other. The results of our present experiments indicate that the expression of the three ribosomal protein genes undergoes independent noncoordinated changes in the large majority of the leukemic populations studied. © 1990, American Association for Cancer Research. All rights reserved.
1990
- Overexpression of the MPO gene occurring in a case of APL without unusual genotypic characteristics
[Articolo su rivista]
Ferrari, S.; Tagliafico, E.; Temperani, P.; Manfredini, R.; Ceccherelli, G.; Zucchini, P.; Tabilio, A.; Donelli, A.; Torelli, G.; Emilia, G.; Torelli, U.
abstract
Northern blot analysis of four typical cases of acute promyelocytic leukemia showed that one of the cell population examined was characterized by a very high level of expression of the myeloperoxidase (MPO) gene. Western blot analysis confirms that the protein content of the cells corresponded to the levels of the MPO mRNA. Southern blot studies of the DNA of this cell population ruled out the presence of any genome amplification or rearrangement. Chromosome hybridization studies in situ confirmed that the MPO gene was translocated on the long arm of chromosome 15. The observation that a typical genomic pattern may or may not be associated with the MPO overexpression leads us to believe that so far it is impossible to reach any conclusion about the significance of the translocation in the genesis of MPO overexpression. © 1990.
1990
- Ratios between the abundance of messenger RNA and the corresponding protein of two growth-related genes, c-myc and vimentin, in leukemia blast cells..
[Articolo su rivista]
Ferrari, S; Tagliafico, Enrico; D'Incá, M; Ceccherelli, G; Manfredini, R; Selleri, L; Donelli, A; Sacchi, Stefano; Torelli, Giuseppe; Torelli, U.
abstract
The abundance of the mRNAs of two growth-related genes, vimentin and c-myc, and that of the corresponding proteins have been studied in unstimulated and phytohemagglutinin-stimulated lymphocytes as well as in 18 populations of leukemic blast cells. The quantitative assay was carried out by densitometric scanning of Northern and Western blots. In normal lymphocytes the mRNA and the protein of both genes were almost undetectable. The phytohemagglutinin stimulation led to a sharp increase of the mRNA and the proteins of vimentin and c-myc. The increase was followed by a progressive fall of the gene products. The rate of decrease of the two mRNAs was similar to that of the corresponding proteins. In some leukemic populations very similar amounts of the vimentin protein were accompanied by amounts of the mRNA differing at least 25 times. Not unlikely, very similar amounts of p62c-myc corresponded to mRNA abundances differing at least 16 times. The coordinated biogenesis of both messenger RNAs and proteins, which occurs in mitogen-stimulated lymphocytes, is substituted, in approximately 30% of the leukemic blast cell populations, by molecular events leading to the accumulation of an excess of mRNA.
1989
- Expression of myeloperoxidase and lactoferrin genes in blast cells of acute lymphoblastic leukemia
[Articolo su rivista]
Manfredini, R.; Tagliafico, E.; Rossi, E.; Ceccherelli, G.
abstract
The myeloperoxidase (MPO) and lactoferrin genes are strictly associated to the granulocytic differentiation pathway. Many reports show that immunologic and molecular cell markers, though to be lineage specific, are in fact expressed in cells of different lineages. We have identified lymphoid blast cell populations expressing the MPO gene at the mRNA level, but lacking any detectable MPO protein. We addressed this issue studying the expression of the MPO and lactoferrin genes in extremely homogenous Acute Lymphoblastic Leukemia (ALL) blast cell populations. The study was carried out by solution hybridization of total cellular mRNA extracted from the leukemic populations with an antisense RNA transcribed by riboprobes. Either MPO or lactoferrin mRNAs were detectable in some ALL populations. We suggest that the expression of these two genes could be a physiologic event in the very early stages of lymphoid cell differentiation.
1988
- Possible correlations between proliferation and differentiation in blast cells of acute promyelocytic leukemia: A molecular study
[Articolo su rivista]
Tagliafico, E.; Manfredini, R.; Selleri, L.; Zucchini, P.; Ceccherelli, G.
abstract
The level of expression of the myeloperoxidase gene (MPO), strictly associated with myeloid differentiation as well as of some cell cycle related genes, such as c-myc and Histone H3, was evaluated by northern blot analysis in blast cells of 4 cases of Acute Promyelocytic Leukemia. MPO gene expression was found to be not related to the growth activity of the cell population. It appears that the differentiation arrest which occurs in the majority of the blast cells is not strictly associated with the proliferation impairment.